Oligomeric compounds and compositions for use in modulation of small non-coding RNAs

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression and function of small non-coding RNAs. The compositions comprise oligomeric compounds, targeted to small non-coding RNAs. Methods of using these compounds for modulation of small non-coding RNAs as well as downstream targets of these RNAs and for diagnosis and treatment of disease associated with small non-coding RNAs are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/909,125 filedJul. 30, 2004, which claims priority to U.S. provisional applicationSer. No. 60/492,056 filed Jul. 31, 2003, Ser. No. 60/516,303 filed Oct.31, 2003, Ser. No. 60/531,596 filed Dec. 19, 2003, and Ser. No.60/562,417 filed Apr. 14, 2004, each which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulationof small non-coding RNAs. In particular, this invention relates tocompounds, particularly oligomeric compounds, which, in someembodiments, hybridize with or sterically interfere with nucleic acidmolecules comprising or encoding small non-coding RNA targets. Suchcompounds are shown herein to modulate the levels of small non-codingRNAs. The oligomeric compounds of the invention may include one or moremodifications thereon resulting in differences in physical or chemicalproperties compared to unmodified nucleic acids. These modifiedoligomeric compounds are used as single compounds or in compositions tomodulate or mimic the targeted nucleic acid comprising or encoding thesmall non-coding RNA. In some embodiments of the invention,modifications include chemical modifications that improve activity ofthe oligomeric compound. In some embodiments, the modifications includemoieties that modify or enhance the pharmacokinetic or pharmacodynamicproperties, stability or nuclease resistance of the oligomeric compound.In some embodiments, the modifications render the oligomeric compoundscapable of sterically interfering with the natural processing of thenucleic acids comprising or encoding the small non-coding RNA targets.

BACKGROUND OF THE INVENTION

RNA genes were once considered relics of a primordial “RNA world” thatwas largely replaced by more efficient proteins. More recently, however,it has become clear that non-coding RNA genes produce functional RNAmolecules with important roles in regulation of gene expression,developmental timing, viral surveillance, and immunity. Not only theclassic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also smallnuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interferingRNAs (siRNAs), tiny non-coding RNAs (tncRNAs), repeat-associated smallinterfering RNAs (rasiRNAs) and microRNAs (miRNAs) are now believed toact in diverse cellular processes such as chromosome maintenance, geneimprinting, pre-mRNA splicing, guiding RNA modifications,transcriptional regulation, and the control of mRNA translation (Eddy,Nat. Rev. Genet., 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003,423, 838-842; Aravin, et al., Dev. Cell, 2003, 5, 337-350). RNA-mediatedprocesses are now also believed to direct heterochromatin formation,genome rearrangements, and DNA elimination (Cerutti, Trends Genet.,2003, 19, 39-46; Couzin, Science, 2002, 298, 2296-2297).

The recently described phenomenon known as RNA interference (RNAi) isinvolves the processing of double stranded RNA into siRNAs by an RNaseIII-like dsRNA-specific enzyme known as Dicer (also known ashelicase-moi) which are then incorporated into a ribonucleoproteincomplex, the RNA-induced silencing complex (RISC). RISC is believed touse the siRNA molecules as a guide to identify complementary RNAs, andan endoribonuclease (to date unidentified) cleaves these target RNAs,resulting in their degradation (Cerutti, Trends Genet., 2003, 19, 39-46;Grishok et al., Cell, 2001, 106, 23-34). In addition to the siRNAs, alarge class of small non-coding RNAs known as microRNAs (miRNAs,originally termed stRNA for “short temporal RNAs”) is believed to play arole in regulation of gene expression employing some of the same playersinvolved in the RNAi pathway (Novina and Sharp, Nature, 2004, 430,161-164).

Like siRNAs, miRNAs are believed to be processed endogenously by theDicer enzyme, and are approximately the same length, and possess thecharacteristic 5′-phosphate and 3′-hydroxyl termini. The miRNAs are alsoincorporated into a ribonucleoprotein complex, the miRNP, which issimilar, and may be identical to the RISC (Bartel and Bartel, PlantPhysiol., 2003, 132, 709-717). More than 200 different miRNAs have beenidentified in plants and animals (Ambros et al., Curr. Biol., 2003, 13,807-818).

In spite of their biochemical and mechanistic similarities, there arealso some differences between siRNAs and miRNAs, based on unique aspectsof their biogenesis. siRNAs are generated from the cleavage of longexogenous or possibly endogenous dsRNA molecules, such as very longhairpins or bimolecular duplexed dsRNA, and numerous siRNAs accumulatefrom both strands of dsRNA precursors. In contrast, mature miRNAs appearto originate from long endogenous primary miRNA transcripts (also knownas pri-miRNAs, pri-mirs or pri-pre-miRNAs) that are often hundreds ofnucleotides in length (Lee, et al., EMBO J., 2002, 21(17), 4663-4670).

The current model of miRNA processing involves primary miRNA transcriptsbeing processed by a nuclear enzyme in the RNase III family known asDrosha, into approximately 70 nucleotide-long pre-miRNAs (also known asstem-loop structures, hairpins, pre-mirs or foldback miRNA precursors)which are subsequently processed by the Dicer RNase into mature miRNAs,approximately 21-25 nucleotides in length. It is believed that, inprocessing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts thepri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′ overhang(Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, PlantPhysiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12; Lee,et al., EMBO J., 2002, 21(17), 4663-4670; Lee, et al., Nature, 2003,425, 415-419). The 3′ two-nucleotide overhang structure, a signature ofRNaseIII cleavage, has been identified as a critical specificitydeterminant in targeting and maintaining small RNAs in the RNAinterference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004,16, 223-9). Both the primary RNA transcripts (pri-miRNAs) and foldbackmiRNA precursors (pre-miRNAs) are believed to be single-stranded RNAmolecules with at least partial double-stranded character, oftencontaining smaller, local internal hairpin structures. Primary miRNAtranscripts may be processed such that one single-stranded mature miRNAmolecule is generated from one arm of the hairpin-like structure of thepri-miRNA. Alternatively, a polycistronic pri-miRNA may contain multiplepre-miRNAs, each processed into a different, single-stranded maturemiRNA.

Naturally occurring miRNAs are characterized by imperfectcomplementarity to their target sequences. Artificially modified miRNAswith sequences completely complementary to their target RNAs have beendesigned and found to function as double stranded siRNAs that inhibitgene expression by reducing RNA transcript levels. Synthetic hairpinRNAs that mimic siRNAs and miRNA precursor molecules were demonstratedto target genes for silencing by degradation and not translationalrepression (McManus et al., RNA, 2002, 8, 842-850).

Tiny non-coding RNA (tncRNA), one class of small non-coding RNAs (Ambroset al., Curr. Biol., 2003, 13, 807-818) produce transcripts similar inlength (20-21 nucleotides) to miRNAs, and are also thought to bedevelopmentally regulated but, unlike miRNAs, tncRNAs are reportedly notprocessed from short hairpin precursors and are not phylogeneticallyconserved. Although none of these tncRNAs are reported to originate frommiRNA hairpin precursors, some are predicted to form potential foldbackstructures reminiscent of pre-miRNAs; these putative tncRNA precursorstructures deviate significantly from those of pre-miRNAs in keycharacteristics, i.e., they exhibit excessive numbers of bulgednucleotides in the stem or have fewer than 16 base pairs involving thesmall RNA (Ambros et al., Curr. Biol., 2003, 13, 807-818).

Recently, another class of small non-coding RNAs, the repeat-associatedsmall interfering RNAs (rasiRNAs) has been isolated from Drosophilamelanogaster. The rasiRNAs are associated with repeated sequences,transposable elements, satellite and microsatellite DNA, and Suppressorof Stellate repeats, suggesting that small RNAs may participate indefining chromatin structure (Aravin, et al., Dev. Cell, 2003, 5,337-350).

A total of 201 different expressed RNA sequences potentially encodingnovel small non-messenger species (smRNAs) has been identified frommouse brain cDNA libraries. Based on sequence and structural motifs,several of these have been assigned to the snoRNA class of nucleolarlocalized molecules known to act as guide RNAs for rRNA modification,whereas others are predicted to direct modification within the U2, U4,or U6 small nuclear RNAs (snRNAs). Some of these newly identified smRNAsremained unclassified and have no identified RNA targets. It wassuggested that some of these RNA species may have novel functionspreviously unknown for snoRNAs, namely the regulation of gene expressionby binding to and/or modifying mRNAs or their precursors via theirantisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953).

To date, the binding and regulatory sites within nucleic acid targets ofthe small non-coding RNAs are largely unknown, although a few putativemotifs have been suggested to exist in the 3′UTR of certain genes (Laiand Posakony, Development, 1997, 124, 4847-4856; Lai, et al.,Development, 2000, 127, 291-306; Lai, Nat. Genet. 2002, 30(4), 363-364).

One miRNA is also believed to act as a cell death regulator, implicatingit in mechanisms of human disease such as cancer. Recently, theDrosophila mir-14 miRNA was identified as a suppressor of apoptotic celldeath and is required for normal fat metabolism. (Xu et al., Curr.Biol., 2003, 13, 790-795).

Downregulation or deletion of other miRNAs has been associated withB-cell chronic lymphocytic leukemia (B-CLL) (Calin et al., Proc. Natl.Acad. Sci. USA, 2002, 99, 15524-15529), and human homologues of themurine mir-143 and mir-145 mature miRNAs were recently reported to beexpressed and processed at reduced steady-state levels at theadenomatous and cancerous stages of colorectal neoplasia (Michael, etal., Mol. Cancer. Res., 2003, 1, 882-891).

Expression of the human mir-30 miRNA specifically blocked thetranslation in human cells of an mRNA containing artificial mir-30target sites. In these studies, putative miRNAs were excised fromtranscripts encompassing artificial miRNA precursors and could inhibitthe expression of mRNAs containing a complementary target site. Thesedata indicate that novel miRNAs can be readily produced in vivo and canbe designed to specifically inactivate the expression of selected targetgenes in human cells (Zeng et al., Mol. Cell, 2002, 9, 1327-1333).

Disclosed and claimed in PCT Publication WO 03/029459 are miRNAs fromseveral species, or a precursor thereof, a nucleotide sequence which isthe complement of said nucleotide sequence which has an identity of atleast 80% to said sequence; and a nucleotide sequence which hybridizesunder stringent conditions to said sequence. Also claimed is apharmaceutical composition containing as an active agent at least one ofsaid nucleic acid and optionally a pharmaceutically acceptable carrier,and a method of identifying microRNA molecules or precursor moleculesthereof comprising ligating 5′- and 3′-adapter molecules to the ends ofa size-fractionated RNA population, reverse transcribing said adaptercontaining RNA population and characterizing the reverse transcriptionproducts (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

Small non-coding RNA-mediated regulation of gene expression is anattractive approach to the treatment of diseases as well as infection bypathogens such as bacteria, viruses and prions and other disordersassociated with RNA expression or processing.

Consequently, there remains a long-felt need for agents that regulategene expression via the mechanisms mediated by small non-coding RNAs.Identification of modified miRNAs or miRNA mimics that can increase ordecrease gene expression or activity is therefore desirable.

The present invention therefore provides oligomeric compounds andmethods useful for modulating gene levels, expression, function orpathways, including those relying on mechanisms of action such as RNAinterference and dsRNA enzymes, as well as antisense and non-antisensemechanisms. One having skill in the art, once armed with this disclosurewill be able, without undue experimentation, to identify compounds,compositions and methods for these uses.

SUMMARY OF THE INVENTION

The present invention provides oligomeric compounds, especially nucleicacid and nucleic acid-like oligomers, which are targeted to or mimicnucleic acids comprising or encoding small non-coding RNAs, and whichact to modulate the levels of small non-coding RNAs, or interfere withtheir function.

The present invention also provides oligomeric compounds comprising afirst strand and a second strand wherein at least one strand contains amodification and wherein a portion of one of the oligomeric compoundstrands is capable of hybridizing to a small non-coding RNA targetnucleic acid.

The present invention also provides oligomeric compounds comprising afirst region and a second region and optionally a third region whereinat least one region contains a modification and wherein a portion of theoligomeric compound is capable of hybridizing to a small non-coding RNAtarget nucleic acid.

The present invention also provides oligomeric compounds, especiallynucleic acid and nucleic acid-like oligomers, which are targeted to anucleic acid encoding human Dicer, and which act to modulate the levelsof the human Dicer RNase III enzyme and interfere with its function, aswell as modulating the levels of small non-coding RNAs.

Pharmaceutical and other compositions comprising the compounds of theinvention are also provided.

Also provided are methods of screening for modulators of smallnon-coding RNAs and methods of modulating the levels of small non-codingRNAs in cells, tissues or animals comprising contacting said cells,tissues or animals with one or more of the compounds or compositions ofthe invention.

Methods of treating an animal, particularly a human, suspected of havingor being prone to a disease or condition associated with expression ofsmall non-coding RNAs are also set forth herein. Such methods compriseoptionally identifying such an animal, and administering atherapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the animal or person.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the interaction of the mir-143 miRNA with three novelbinding sites in the ERK5 mRNA coding sequence (GenBank AccessionNM_(—)139032.1) identified herein, along with their bimolecularhybridization free energies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides oligomeric compounds useful in, forexample, the modulation of expression, endogenous levels or the functionof small non-coding RNAs. As used herein, the term “small non-codingRNA” is used to encompass, without limitation, a polynucleotide moleculeranging from about 17 to about 450 nucleotides in length, which can beendogenously transcribed or produced exogenously (chemically orsynthetically), but is not translated into a protein. Small non-codingRNAs may include isolated single-, double-, or multiple-strandedmolecules, any of which may include regions of intrastrand nucleobasecomplementarity, said regions capable of folding and forming a moleculewith fully or partially double-stranded or multiple-stranded characterbased on regions of perfect or imperfect complementarity. Examples ofsmall non-coding RNAs include, but are not limited to, primary miRNAtranscripts (also known as pri-pre-miRNAs, pri-mirs and pri-miRNAs,which range from around 70 nucleotides to about 450 nucleotides inlength and often taking the form of a hairpin structure); pre-miRNAs(also known as pre-mirs and foldback miRNA precursors, which range fromaround 50 nucleotides to around 110 nucleotides in length); miRNAs (alsoknown as microRNAs, Mirs, miRs, mirs, and mature miRNAs, and generallyrefer either to double-stranded intermediate molecules around 17 toabout 25 nucleotides in length, or to single-stranded miRNAs, which maycomprise a bulged structure upon hybridization with a partiallycomplementary target nucleic acid molecule); or mimics of pri-miRNAs,pre-miRNAs or miRNAs. Small non-coding RNAs can be endogenouslytranscribed in cells, or can be synthetic oligonucleotides, in vitrotranscribed polynucleotides or nucleic acid oligomeric compoundsexpressed from vectors. Pri-miRNAs and pre-miRNAs, or mimics thereof,may be processed into smaller molecules.

As used herein, the term “miRNA precursor” is used to encompass, withoutlimitation, primary RNA transcripts, pri-miRNAs and pre-miRNAs.

In some embodiments, pri-miRNAs, or mimics thereof, are 70 to 450nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449 or 450 nucleobases in length, or any rangetherewithin.

In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 430nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 nucleobases inlength, or any range therewithin.

In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 280nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280nucleobases in length, or any range therewithin.

In some embodiments, pre-miRNAs, or mimics thereof, are 50 to 110nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 7172, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109 or 110 nucleobases in length, or any rangetherewithin. In some embodiments, pre-miRNAs, or mimics thereof, are 60to 80 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80nucleobases in length, or any range therewithin.

In some embodiments, miRNAs, or mimics thereof, are 15 to 49 nucleobasesin length. One having ordinary skill in the art will appreciate thatthis embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleobases in length, or any rangetherewithin. In some embodiments, miRNAs, or mimics thereof, are 17 to25 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 17, 18, 19, 20,21, 22, 23, 24 or 25 nucleobases in length, or any range therewithin.

Oligomeric compounds of the invention modulate the levels, expression orfunction of small non-coding RNAs by hybridizing to a nucleic acidcomprising or encoding a small non-coding RNA nucleic acid targetresulting in alteration of normal function by, for example, facilitatingdestruction of the small non-coding RNA through cleavage, bysequestration, or by sterically occluding the function of the smallnon-coding RNA. Further, modified synthetic oligomeric compounds of thepresent invention may be designed to mimic endogenous small non-codingRNAs. These modifications include, but are not limited to, improvedpharmacokinetic or pharmacodynamic properties, binding affinity,stability, charge, localization or uptake. Synthetic mimics cantherefore act as replacements for small non-coding RNAs, as competitiveinhibitors of naturally occurring small non-coding RNAs or as deliverysystems wherein the mimic construct contains one or more functionalcomponents.

As used herein, the terms “target nucleic acid,” “target RNA,” “targetRNA transcript” or “nucleic acid target” are used to encompass anynucleic acid capable of being targeted including, without limitation,RNA (including microRNAs, stRNAs, small nuclear RNAs, small nucleolarRNAs, small ribosomal RNAs, small hairpin RNAs, endogenous antisenseRNAs, guide RNAs, tiny noncoding RNAs, small single or double strandedRNAs that are encoded by heterochromatic repeats at centromeres or otherchromosomal origin, and any precursors thereof). These nucleic acidtargets can be coding or non-coding sequences; pre-mRNAs or mRNAs;single- or double-stranded, or single-stranded with partialdouble-stranded character; may occur naturally within introns or exonsof messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), or transfer RNAs(tRNAs); and can be endogenously transcribed or exogenously produced.

In some embodiments of this invention, modulation of small non-codingRNA levels, expression or function is achieved via oligomeric compoundswhich target a further RNA associated with the particular smallnon-coding RNA. This association can be a physical association betweenthat RNA and the particular small non-coding RNA such as, but notlimited to, in an RNA or ribonucleoprotein complex. This association canalso be within the context of a biological pathway, such as but notlimited to, the regulation of levels, expression or function of aprotein-encoding mRNA or its precursor by a small non-coding RNA. Assuch, the invention provides for modulation of the levels, expression orfunction of a target nucleic acid where the target nucleic acid is amessenger RNA whose expression levels and/or function are associatedwith one or more small non-coding RNAs. The messenger RNA function orprocessing may be disrupted by degradation through an antisensemechanism, including but not limited to, RNA interference, or RNase H,as well as other mechanisms wherein double stranded nucleic acidstructures are recognized and degraded, cleaved, sterically occluded,sequestered or otherwise rendered inoperable.

The compounds or compositions of the present invention may alsointerfere with the function of endogenous RNA molecules. The functionsof RNA to be interfered with can include, for example, nuclear eventssuch as replication or transcription as the compounds of the presentinvention could target or mimic small non-coding RNAs in these cellularprocesses. Replication and transcription, for example, can be from anendogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includecytoplasmic events such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, RNA signalingand regulatory activities, and catalytic activity or complex formationinvolving the RNA which may be engaged in or facilitated by the RNA asthe compounds of the present invention could target or mimic smallnon-coding RNAs in these cellular processes.

In the context of the present invention, “modulation” and “modulation ofexpression” mean either an increase (stimulation) or a decrease(inhibition) in the amount or levels of a small non-coding RNA, nucleicacid target, an RNA or protein associated with a small non-coding RNA,or a downstream target of the small non-coding RNA (e.g., a mRNArepresenting a protein-coding nucleic acid that is regulated by a smallnon-coding RNA). Inhibition is a suitable form of modulation and smallnon-coding RNA is a suitable target nucleic acid.

In the context of the present invention, “modulation of function” meansan alteration in the function of the small non-coding RNA or analteration in the function of any cellular component with which thesmall non-coding RNA has an association or downstream effect.

The present invention provides, inter alia, oligomeric compounds andcompositions containing the same wherein the oligomeric compoundincludes one or more modifications that render the compound capable ofsupporting modulation of the levels, expression or function of the smallnon-coding RNA by a degradation or cleavage mechanism.

The present invention also provides methods of maintaining a pluripotentstem cell comprising contacting the cell with an effective amount of anoligomeric compound targeting human Dicer. The pluripotent stem cell canbe present in a sample of cord blood or bone marrow, or may be presentas part of a cell line. In addition, the pluripotent stem cell can be anembryonic stem cell.

The present invention also provides oligomeric compounds andcompositions containing the same wherein the oligomeric compoundincludes one or more modifications that render the compound capable ofblocking or interfering with the levels, expression or function of oneor more small non-coding RNAs by steric occlusion.

The present invention also provides oligomeric compounds andcompositions containing the same wherein the oligomeric compoundincludes one or more modifications or structural elements or motifs thatrender the compound capable of mimicking or replacing one or more smallnon-coding RNAs.

Oligomeric Compounds

In the context of the present invention, the term “oligomericcompound(s)” refers to polymeric structures which are capable ofhybridizing to at least a region of a small non-coding RNA molecule or atarget of small non-coding RNAs, or polymeric structures which arecapable of mimicking small non-coding RNAs. The term “oligomericcompound” includes, but is not limited to, compounds comprisingoligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics and combinations of these. Oligomeric compoundsalso include, but are not limited to, antisense oligomeric compounds,antisense oligonucleotides, siRNAs, alternate splicers, primers, probesand other compounds that hybridize to at least a portion of the targetnucleic acid. Oligomeric compounds are routinely prepared linearly butcan be joined or otherwise prepared to be circular and may also includebranching. Separate oligomeric compounds can hybridize to form doublestranded compounds that can be blunt-ended or may include overhangs onone or both termini. In general, an oligomeric compound comprises abackbone of linked monomeric subunits where each linked monomericsubunit is directly or indirectly attached to a heterocyclic basemoiety. The linkages joining the monomeric subunits, the sugar moietiesor sugar surrogates and the heterocyclic base moieties can beindependently modified giving rise to a plurality of motifs for theresulting oligomeric compounds including hemimers, gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond. In addition, linear compounds may haveinternal nucleobase complementarity and may therefore fold in a manneras to produce a fully or partially double-stranded structure. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside linkages of the oligonucleotide. Thenormal internucleoside linkage of RNA and DNA is a 3′ to 5′phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refersgenerally to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA). This term includes oligonucleotidescomposed of naturally occurring nucleobases, sugars and covalentinternucleoside linkages. The term “oligonucleotide analog” refers tooligonucleotides that have one or more non-naturally occurring portionswhich function in a similar manner to oligonucleotides. Suchnon-naturally occurring oligonucleotides are often selected overnaturally occurring forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for otheroligonucleotides or nucleic acid targets and increased stability in thepresence of nucleases.

In the context of this invention, the term “oligonucleoside” refers tonucleosides that are joined by internucleoside linkages that do not havephosphorus atoms. Internucleoside linkages of this type include shortchain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatomcycloalkyl, one or more short chain heteroatomic and one or more shortchain heterocyclic. These internucleoside linkages include but are notlimited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl,thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl,sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide,amide and others having mixed N, O, S and CH₂ component parts. Inaddition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodifications. Additional nucleosides amenable to the present inventionhaving altered base moieties and or altered sugar moieties are disclosedin U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323.

For nucleotides that are incorporated into oligonucleotides of theinvention, these nucleotides can have sugar portions that correspond tonaturally occurring sugars or modified sugars. Representative modifiedsugars include carbocyclic or acyclic sugars, sugars having substituentgroups at one or more of their 2′, 3′ or 4′ positions and sugars havingsubstituents in place of one or more hydrogen atoms of the sugar.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigomeric compounds are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified oligonucleotides. All such oligomericcompounds are comprehended by this invention so long as they functioneffectively to mimic the structure or function of a desired RNA or DNAoligonucleotide strand.

A class of representative base modifications include tricyclic cytosineanalog, termed “G clamp” (Lin, et at., J. Am. Chem. Soc. 1998, 120,8531). This analog can form four hydrogen bonds with a complementaryguanine (G) by simultaneously recognizing the Watson-Crick and Hoogsteenfaces of the targeted G. This G clamp modification when incorporatedinto phosphorothioate oligomeric compounds, dramatically enhancespotencies as measured by target reduction in cell culture. Theoligomeric compounds of the invention also can includephenoxazine-substituted bases of the type disclosed by Flanagan, et al.,Nat. Biotechnol. 1999, 17(1), 48-52.

The oligomeric compounds in accordance with this invention comprise fromabout 8 to about 80 monomeric subunits (i.e. from about 8 to about 80linked nucleosides). One of ordinary skill in the art will appreciatethat the invention embodies oligomeric compounds of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits inlength, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 12 to50 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 13 to80 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or anyrange therewithin.

In one embodiment, the oligomeric compounds of the invention are 15 to30 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 subunits inlength, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 70 to450 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449 or 450 subunits in length, or anyrange therewithin.

In one embodiment, the oligomeric compounds of the invention are 110 to430 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419,420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 subunits inlength, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 110 to280 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or280 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 50 to110 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70,71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109 or 110 subunits in length, or any rangetherewithin.

In one embodiment, the oligomeric compounds of the invention are 60 to80 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or80 subunits in length, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 15 to49 monomeric subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 subunits inlength, or any range therewithin.

In one embodiment, the oligomeric compounds of the invention are 17 to25 subunits in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 17, 18, 19, 20,21, 22, 23, 24 or 25 subunits in length, or any range therewithin.

In accordance with the present invention, oligomeric compounds designedto mimic pri-miRNAs are from about 70 to about 450 monomeric subunits inlength, or from about 110 to 430 subunits in length. Oligomericcompounds of the invention designed to mimic pre-miRNAs are from about50 to about 110 monomeric subunits in length, or from about 60 to about80 subunits in length. Oligomeric compounds of the invention designed tomimic mature miRNAs are from about 17 to about 25 monomeric subunits inlength, and can be single- or double-stranded with either or bothstrands comprising from about 17 to about 25 subunits.

As used herein, the term “about” means±5% of the variable thereafter.

The size or length of any oligomeric compound of the present invention,within any range cited herein, can be determined as follows:

Let R(m, n+m−1) be a region from a target nucleobase sequence, where “n”is the 5′-most nucleobase position of the region, where “n+m−1” is the3′-most nucleobase position of the region and where “m” is the length ofthe region. A set “S(m)”, of regions of length “m” is defined as theregions where n ranges from 1 to L−m+1, where L is the length of thetarget nucleic acid sequence and L>m. A set, “A”, of all regions can beconstructed as a union of the sets of regions for each length from wherem is greater than or equal to a lower limit of any recited range (8 inthis example) and is less than or equal to the upper limit of anyrecited range (80 in this example).

This set of regions can be represented using the following mathematicalnotation:

$A =  {{\bigcup\limits_{m}{{S(m)}\mspace{14mu}{where}\mspace{14mu} m}} \in N} \middle| {8 \leq m \leq 80} $and S(m) = {R_(n, n + m − 1)|n ∈ {1, 2, 3, …  , L − m + 1}}

where the mathematical operator | indicates “such that”,

where the mathematical operator ε indicates “a member of a set” (e.g.yεZ indicates that element y is a member of set Z),

where x is a variable,

where N indicates all natural numbers, defined as positive integers,

and where the mathematical operator ∪ indicates “the union of sets”.

For example, the set of regions for m equal to 8, 20 and 80 can beconstructed in the following manner. The set of regions, each 8monomeric subunits in length, S(m=8), in a target nucleic acid sequence100 subunits in length (L=100), beginning at position 1 (n=1) of thetarget nucleic acid sequence, can be created using the followingexpression:S(8)={R _(1,8) |nε{1, 2, 3, . . . , 93}}and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10,4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21,15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31,25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41,35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51,45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61,55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71,65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81,75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91,85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 monomeric subunits in length, in atarget sequence 100 subunits in length, beginning at position 1 of thetarget nucleic acid sequence, can be described using the followingexpression:S(20)={R _(1,20) |nε{1, 2, 3, . . . , 81}}and describes the set of regions comprising nucleobases 1-20, 2-21,3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32,14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42,24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52,34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62,44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72,54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82,64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92,74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 monomeric subunits in length, in atarget sequence 100 subunits in length, beginning at position 1 of thetarget nucleic acid sequence, can be described using the followingexpression:S(80)={R _(1,80) |nε{1, 2, 3, . . . , 21}}and describes the set of regions comprising nucleobases 1-80, 2-81,3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92,14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

The union of these aforementioned example sets and other sets forlengths from 10 to 19 and 21 to 79 can be described using themathematical expression

$A = {\bigcup\limits_{m}{S(m)}}$

where ∪ represents the union of the sets obtained by combining allmembers of all sets.

Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . .93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The mathematical expressions described herein define all possible targetregions in a target nucleic acid sequence of any length L, where theregion is of length m, and where m is greater than or equal to the lowerlimit and less than or equal to the upper limit of monomeric units, andwhere m is less than L, and where n is less than L−m+1.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases that pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An oligomeric compound of the invention is “specifically hybridizable”when association of the compound with the target nucleic acid interfereswith the normal function of the target nucleic acid to alter theactivity, disrupt the function, or modulate the level of the targetnucleic acid, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligomeric compound to non-targetnucleic acid sequences under conditions in which specific hybridizationis desired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and under standard assay conditions inthe case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention; “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated. One having ordinary skill in the art willunderstand variability in the experimental protocols and be able todetermine when conditions are optimal for stringent hybridization withminimal non-specific hybridization events.

“Complementary,” as used herein, refers to the capacity for precisepairing of two monomeric subunits regardless of where in the oligomericcompound or target nucleic acid the two are located. For example, if amonomeric subunit at a certain position of an oligomeric compound iscapable of hydrogen bonding with a monomeric subunit at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligomeric compound and the target nucleic acid isconsidered to be a complementary position. The oligomeric compound andthe target nucleic acid are “substantially complementary” to each otherwhen a sufficient number of complementary positions in each molecule areoccupied by monomeric subunits that can hydrogen bond with each other.Thus, the term “substantially complementary” is used to indicate asufficient degree of precise pairing over a sufficient number ofmonomeric subunits such that stable and specific binding occurs betweenthe oligomeric compound and a target nucleic acid.

Generally, an oligomeric compound is “antisense” to a target nucleicacid when, written in the 5′ to 3′ direction, it comprises the reversecomplement of the corresponding region of the target nucleic acid.“Antisense compounds” are also often defined in the art to comprise thefurther limitation of, once hybridized to a target, being able to induceor trigger a reduction in target gene expression.

It is understood in the art that the sequence of the oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization (e.g., a bulge, a loopstructure or a hairpin structure).

In some embodiments of the invention, the oligomeric compounds compriseat least 50%, at least 60%, at least 70%, at least 75%, at least 80%, orat least 85% sequence complementarity to a target region within thetarget nucleic acid. In other embodiments of the invention, theoligomeric compounds comprise at least 90% sequence complementarity to atarget region within the target nucleic acid. In other embodiments ofthe invention, the oligomeric compounds comprise at least 95% or atleast 99% sequence complementarity to a target region within the targetnucleic acid. For example, an oligomeric compound in which 18 of 20nucleobases of the oligomeric compound are complementary to a targetsequence would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an oligomericcompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anoligomeric compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

In some embodiments of the invention, the oligomeric compounds act asmimics or replacements for small non-coding RNAs. In this case, theoligomeric compounds of the invention can comprise at least 70% sequenceidentity to a small non-coding RNA or a region thereof. In someembodiments the oligomeric compounds of the invention can comprise atleast 90% sequence identity and in some embodiments can comprise atleast 95% sequence identity to a small non-coding RNA or a regionthereof.

“Targeting” an oligomeric compound to a particular nucleic acidmolecule, in the context of this invention, can be a multistep process.The process usually begins with the identification of a target nucleicacid whose levels, expression or function is to be modulated. Thistarget nucleic acid may be, for example, a mRNA transcribed from acellular gene whose expression is associated with a particular disorderor disease state, a small non-coding RNA or its precursor, or a nucleicacid molecule from an infectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe interaction to occur such that the desired effect, e.g., modulationof levels, expression or function, will result. Within the context ofthe present invention, the term “region” is defined as a portion of thetarget nucleic acid having at least one identifiable sequence,structure, function, or characteristic. Within regions of target nucleicacids are segments. “Segments” are defined as smaller or sub-portions ofregions within a target nucleic acid. “Sites,” as used in the presentinvention, are defined as specific positions within a target nucleicacid. The terms region, segment, and site can also be used to describean oligomeric compound of the invention such as for example a gappedoligomeric compound having three separate segments.

Targets of the present invention include both coding and non-codingnucleic acid sequences. For coding nucleic acid sequences, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon.” A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA transcribed from a gene encoding anucleic acid target, regardless of the sequence(s) of such codons. It isalso known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the oligomeric compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, afurther suitable region is the intragenic region encompassing thetranslation initiation or termination codon of the open reading frame(ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsosuitable to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also target sites. mRNA transcripts produced via the process ofsplicing of two (or more) mRNAs from different gene sources are known as“fusion transcripts.” It is also known that introns can be effectivelytargeted using oligomeric compounds targeted to, precursor molecules forexample, pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants.” More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequences.

Upon excision of one or more exon or intron regions, or portionsthereof, during splicing, pre-mRNA variants produce smaller “mRNAvariants.” Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants.” If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso target nucleic acids.

Certain non-coding RNA genes are known to produce functional RNAmolecules with important roles in diverse cellular processes. Suchnon-translated, non-coding RNA molecules can include ribosomal RNAs,tRNAs, snRNAs, snoRNAs, tncRNAs, rasiRNAs, short hairpin RNAs (shRNAs),short temporal RNAs (stRNAs), short hairpin RNAs (shRNAs), siRNAs,miRNAs and smnRNAs. These non-coding RNA genes and their products arealso suitable targets of the compounds of the invention. Such cellularprocesses include transcriptional regulation, translational regulation,developmental timing, viral surveillance, immunity, chromosomemaintenance, ribosomal structure and function, gene imprinting,subcellular compartmentalization, pre-mRNA splicing, and guidance of RNAmodifications. RNA-mediated processes are now also believed to directheterochromatin formation, genome rearrangements, cellulardifferentiation and DNA elimination.

A total of 201 different expressed RNA sequences potentially encodingnovel small non-messenger species (smnRNAs) has been identified frommouse brain cDNA libraries. Based on sequence and structural motifs,several of these have been assigned to the snoRNA class of nucleolarlocalized molecules known to act as guide RNAs for rRNA modification,whereas others are predicted to direct modification within the U2, U4,or U6 small nuclear RNAs (snRNAs). Some of these newly identifiedsmnRNAs remained unclassified and have no identified RNA targets. It wassuggested that some of these RNA species may have novel functionspreviously unknown for snoRNAs, namely the regulation of gene expressionby binding to and/or modifying mRNAs or their precursors via theirantisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953).Therefore, these smRNAs are also suitable targets for the compounds ofthe present invention.

The locations on the target nucleic acid to which compounds andcompositions of the invention hybridize are herein referred to as“suitable target segments.” As used herein the term “suitable targetsegment” is defined as at least an 8-nucleobase portion of a targetregion to which oligomeric compound is targeted.

Once one or more targets, target regions, segments or sites have beenidentified, oligomeric compounds are designed to be sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect. The desired effectmay include, but is not limited to modulation of the levels, expressionor function of the target.

In accordance with the present invention, a series of single strandedoligomeric compounds can be designed to target or mimic one or morespecific small non-coding RNAs. These oligomeric compounds can be of aspecified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to30, 70 to 450, 110 to 430, 110 to 280, 50 to 110, 60 to 80, 15 to 49, 17to 25 or 19 to 23 nucleotides long and have one or more modifications.

In accordance with one embodiment of the invention, a series ofdouble-stranded oligomeric compounds (duplexes) comprising, as theantisense strand, the single-stranded oligomeric compounds of thepresent invention, and the fully or partially complementary sensestrand, can be designed to modulate the levels, expression or functionof one or more small non-coding RNAs or small non-coding RNA targets.One or both termini of the duplex strands may be modified by theaddition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the duplex may be designed and synthesizedas the complement of the antisense strand and may also containmodifications or additions to either terminus. For example, in oneembodiment, both strands of the duplex would be complementary over thecentral region of the duplex, each having overhangs at one or bothtermini.

For the purposes of this invention, the combination of an antisensestrand and a sense strand, each of which can be of a specified length,for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 15 to 49, 17 to25 or 19 to 23 subunits long, is identified as a complementary pair ofoligomeric compounds. This complementary pair of oligonucleotides caninclude additional nucleotides on either of their 5′ or 3′ ends. Theycan include other molecules or molecular structures on their 3′ or 5′ends, such as a phosphate group on the 5′ end, or non-nucleic acidmoieties conjugated to either terminus of either strand or both strands.One group of compounds of the invention includes a phosphate group onthe 5′ end of the antisense strand compound. Other compounds alsoinclude a phosphate group on the 5′ end of the sense strand compound.Some compounds include additional nucleotides such as a two baseoverhang on the 3′ end as well as those lacking overhangs.

For example, a complementary pair of oligomeric compounds may comprisean antisense strand oligomeric compound having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO:2181), having a two-nucleobase overhangof deoxythymidine (dT) and its complement sense strand. Thiscomplementary pair of oligomeric compounds would have the followingstructure:

In some embodiments, a single-stranded oligomeric compound may bedesigned comprising the antisense portion as a first region and thesense portion as a second region. The first and second regions can belinked together by either a nucleotide linker (a string of one or morenucleotides that are linked together in a sequence) or by anon-nucleotide linker region or by a combination of both a nucleotideand non-nucleotide structure. In any of these structures, the oligomericcompound, when folded back on itself, would form at least a partiallycomplementary structure at least between a portion of the first region,the antisense portion, and a portion of the second region, the senseportion.

In one embodiment, the invention includes an oligomeric compound/proteincomposition. This composition has both an oligomeric compound componentand a protein component. The oligomeric compound component comprises atleast one oligomeric compound, either the antisense or the senseoligomeric compound but preferably the antisense oligomeric compound(the oligomeric compound that is antisense to the target nucleic acid).The protein component of the composition comprises at least one proteinthat forms a portion of the RNA-induced silencing complex, i.e., theRISC complex. The oligomeric compound component can also comprise bothantisense and sense strand oligomeric compounds.

RISC is a ribonucleoprotein complex that contains proteins of theArgonaute family of proteins. While not wishing to be bound by theory,it is believed that the Argonaute proteins are a class of proteins, someof which have been shown to contain a PAZ and/or a Piwi domain and thathave been implicated in processes previously linked toposttranscriptional silencing. The Argonaute family of proteinsincludes, but depending on species, is not necessary limited to e1F2C1and e1F2C2. It is also believed that at least the antisense strand ofdouble-stranded compounds shown to act as siRNAs is bound to one of theprotein components that form the RISC complex, and that the RISC complexinteracts with the ribosomes or polyribosome complexes which may containsmall non-coding RNA molecules amenable to targeting with the oligomericcompounds of the present invention. Consequently, one embodiment of theinvention includes oligomeric compounds that mimic RNA components of theRISC complex.

In one embodiment, the oligomeric compounds of the invention aredesigned to exert their modulatory effects via mimicking or targetingsmall non-coding RNAs associated with cellular factors such astransporters or chaperones. These cellular factors can be protein, lipidor carbohydrate based and can have structural or enzymatic functionsthat may or may not require the complexation of one or more metal ions.

Furthermore, the oligomeric compounds of the invention can have one ormore moieties bound or conjugated, which facilitates the active orpassive transport, localization, or compartmentalization of theoligomeric compound. Cellular localization includes, but is not limitedto, localization to within the nucleus, the nucleolus, or the cytoplasm.Compartmentalization includes, but is not limited to, any directedmovement of the oligonucleotides of the invention to a cellularcompartment including the nucleus, nucleolus, mitochondrion, orimbedding into a cellular membrane.

In some embodiments of the invention, the oligomeric compounds aredesigned to exert their modulatory effects via mimicking or targetingsmall non-coding RNAs associated with cellular factors that affect geneexpression, more specifically those involved in RNA or DNAmodifications. These modifications include, but are not limited to,posttranscriptional or chromosomal modifications such as methylation,acetylation, pseudouridylation or amination.

Furthermore, the oligomeric compounds of the invention comprise one ormore conjugate moieties which facilitate posttranscriptionalmodification.

The oligomeric compounds of the invention may be in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may elicit the action of one or more enzymesor proteins to effect modulation of the levels, expression or functionof the target nucleic acid.

One non-limiting example of such a protein is the Drosha RNase IIIenzyme. Drosha is a nuclear enzyme that processes long primary RNAtranscripts (pri-miRNAs) from approximately 70 to 450 nucleotides inlength into pre-miRNAs (from about 50 to about 80 nucleotides in length)which are exported from the nucleus to encounter the human Dicer enzymewhich then processes pre-miRNAs into miRNAs. It is believed that, inprocessing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts thepri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′ overhang(Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotideoverhang structure, a signature of RNaseIII enzymatic cleavage, has beenidentified as a critical specificity determinant in targeting andmaintaining small RNAs in the RNA interference pathway (Murchison, etal., Curr. Opin. Cell Biol., 2004, 16, 223-9).

A further non-limiting example involves the enzymes of the RISC complex.Use of the RISC complex to effect cleavage of RNA targets therebygreatly enhances the efficiency of oligonucleotide-mediated inhibitionof gene expression. Similar roles have been postulated for otherribonucleases such as those in the RNase III and ribonuclease L familyof enzymes.

Oligomeric compounds or compositions of the invention are used to inducepotent and specific modulation of gene function through interactionswith or mimicry of small non-coding RNAs that are processed by the RISCcomplex. These compounds include single-stranded oligomeric compoundsthat bind in a RISC complex, double-stranded antisense/sense pairs ofoligomeric compounds, or single-stranded oligomeric compounds thatinclude both an antisense portion and a sense portion.

General Oligomer Synthesis:

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA like compounds (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al.,Applications of Chemically synthesized RNA in RNA:Protein Interactions,Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713)synthesis as appropriate. In addition, specific protocols for thesynthesis of oligomeric compounds of the invention are illustrated inthe examples below.

RNA oligomers can be synthesized by methods disclosed herein orpurchased from various RNA synthesis companies such as for exampleDharmacon Research Inc., (Lafayette, Colo.).

Irrespective of the particular protocol used, the oligomeric compoundsused in accordance with this invention may be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed.

Synthesis of Nucleoside Phosphoramidites:

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—(N,Ndimethylaminooxyethyl)-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite),2′-(Aminooxyethoxy) nucleoside amidites,N²-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite),2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Oligonucleotide and Oligonucleoside Synthesis:

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.Nos. 5,256,775 or 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages are prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289,all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

RNA Synthesis:

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett, 1994, 25, 4311-4314; Wincott, F.et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron,1967, 23, 2315-2331).

The present invention is also useful for the preparation of oligomericcompounds incorporating at least one 2′-O-protected nucleoside. Afterincorporation and appropriate deprotection the 2′-O-protected nucleosidewill be converted to a ribonucleoside at the position of incorporation.The number and position of the 2-ribonucleoside units in the finaloligomeric compound can vary from one at any site or the strategy can beused to prepare up to a full 2′-OH modified oligomeric compound. All2′-O-protecting groups amenable to the synthesis of oligomeric compoundsare included in the present invention.

In general a protected nucleoside is attached to a solid support by forexample a succinate linker. Then the oligonucleotide is elongated byrepeated cycles of deprotecting the 5′-terminal hydroxyl group, couplingof a further nucleoside unit, capping and oxidation (alternativelysulfurization). In a more frequently used method of synthesis thecompleted oligonucleotide is cleaved from the solid support with theremoval of phosphate protecting groups and exocyclic amino protectinggroups by treatment with an ammonia solution. Then a furtherdeprotection step is normally required for the more specializedprotecting groups used for the protection of 2′-hydroxyl groups whichwill give the fully deprotected oligonucleotide.

A large number of 2′-O-protecting groups have been used for thesynthesis of oligoribonucleotides but over the years more effectivegroups have been discovered. The key to an effective 2′-O-protectinggroup is that it is capable of selectively being introduced at the2′-O-position and that it can be removed easily after synthesis withoutthe formation of unwanted side products. The protecting group also needsto be inert to the normal deprotecting, coupling, and capping stepsrequired for oligoribonucleotide synthesis. Some of the protectinggroups used initially for oligoribonucleotide synthesis includedtetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groupsare not compatible with all 5′-O-protecting groups so modified versionswere used with 5′-DMT groups such as1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identifieda number of piperidine derivatives (like Fpmp) that are useful in thesynthesis of oligoribonucleotides including1-((chloro-4-methyl)phenyl)-4′-methoxypiperidin-4-yl (Reese et al,Tetrahedron Lett., 1986, (27), 2291). Another approach was to replacethe standard 5′-DMT (dimethoxytrityl) group with protecting groups thatwere removed under non-acidic conditions such as levulinyl and9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile2′-protecting groups for oligoribonucleotide synthesis. Another morewidely used protecting group initially used for the synthesis ofoligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al,Tetrahedron Lett., 1974, 2861; Hakimelahi et al, Tetrahedron Lett.,1981, (22), 2543; and Jones et at., J. Chem. Soc. Perkin I., 2762). The2′-O-protecting groups can require special reagents for their removalsuch as for example the t-butyldimethylsilyl group is normally removedafter all other cleaving/deprotecting steps by treatment of theoligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups(Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoridelabile and photolabile protecting groups that are removed using moderateconditions. One photolabile group that was examined was the(2-(nitrobenzyl)oxy)methyl (nbm) protecting group (Schwartz et al,Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examinedincluded a number structurally related formaldehyde acetal-derived,2′-O-protecting groups. Also prepared were a number of relatedprotecting groups for preparing 2′-O-alkylated nucleosidephosphoramidites including 2′-O-((triisopropylsilyl)oxy)methyl(2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was preparedto be used orthogonally to the TOM group was2′-O-((R)-1-(2-nitrophenyl)ethyloxy)methyl) ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acidlabile) and an acid labile 2′-O-protecting group has been reported(Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number ofpossible silyl ethers were examined for 5′-O-protection and a number ofacetals and orthoesters were examined for 2′-O-protection. Theprotection scheme that gave the best results was 5′-O-silyl ether-2′-ACE(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether(DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses amodified phosphoramidite synthesis approach in that some differentreagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis ofoligoribonucleotides the main RNA synthesis strategies that arepresently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP),2′-O-((triisopropylsilyl)oxy)methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. Such an activator would also be amenable to the presentinvention.

The structures corresponding to these protecting groups are shown below.

TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;

TOM=2′-O-((triisopropylsilyl)oxy)methyl;

DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether-2′-O-bis(2-acetoxyethoxy)methyl

FPMP=5′-O-DMT-2′-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl)

All of the aforementioned RNA synthesis strategies are amenable to thepresent invention. Strategies that would be a hybrid of the above e.g.using a 5′-protecting group from one strategy with a 2′-O-protectingfrom another strategy is also amenable to the present invention.

The preparation of ribonucleotides and oligomeric compounds having atleast one ribonucleoside incorporated and all the possibleconfigurations falling in between these two extremes are encompassed bythe present invention. The corresponding oligomeric compounds can behybridized to further oligomeric compounds includingoligoribonucleotides having regions of complementarity to formdouble-stranded (duplexed) oligomeric compounds.

The methods of preparing oligomeric compounds of the present inventioncan also be applied in the areas of drug discovery and targetvalidation.

Oligonucleotide Isolation:

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et at.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Oligonucleotide Synthesis—96 Well Plate Format:

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Oligonucleotide Analysis—96-Well Plate Format:

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates were diluted fromthe master plate using single and multi-channel robotic pipettors.Plates were judged to be acceptable if at least 85% of the oligomericcompounds on the plate were at least 85% full length.

For double-stranded compounds of the invention, once synthesized, thecomplementary strands are annealed. The single strands are aliquoted anddiluted to a concentration of 50 μM. Once diluted, 30 μL of each strandis combined with 15 μL of a 5× solution of annealing buffer. The finalconcentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, and 2 mM magnesium acetate. The final volume is 75 μL. Thissolution is incubated for 1 minute at 90° C. and then centrifuged for 15seconds. The tube is allowed to sit for 1 hour at 37° C. at which timethe double-stranded compounds are used in experimentation. The finalconcentration of the duplexed compound is 20 μM. This solution can bestored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the double-stranded compounds are evaluated for theirability to modulate target levels, expression or function. When cellsreach 80% confluency, they are treated with synthetic double-strandedcompounds comprising at least one oligomeric compound of the invention.For cells grown in 96-well plates, wells are washed once with 200 μLOPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130μL of OPTI-MEM™-1 containing 12 μg/mL LIPOFECTIN™ (InvitrogenCorporation, Carlsbad, Calif.) and the desired double stranded compoundat a final concentration of 200 nM. After 5 hours of treatment, themedium is replaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby real-time RT-PCR.

Specific examples of oligomeric compounds useful in this inventioninclude oligonucleotides containing modified e.g. non-naturallyoccurring internucleoside linkages. As defined in this specification,oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage(phosphorothioate) did not significantly interfere with RNAi activity.Based on this observation, it is suggested that certain oligomericcompounds of the invention can also have one or more modifiedinternucleoside linkages. A suitable phosphorus-containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage.

Modified oligonucleotide backbones (internucleoside linkages) containinga phosphorus atom therein include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In other embodiments of the invention, oligomeric compounds have one ormore phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Amideinternucleoside linkages are disclosed in the above referenced U.S. Pat.No. 5,602,240.

Modified oligonucleotide backbones (internucleoside linkages) that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Another group of oligomeric compounds amenable to the present inventionincludes oligonucleotide mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligomeric compounds whereinonly the furanose ring or both the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.One such oligomeric compound, an oligonucleotide mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA oligomeric compounds, thesugar-backbone of an oligonucleotide is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA oligomeric compounds include,but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and5,719,262, each of which is herein incorporated by reference. Teachingof PNA oligomeric compounds can be found in Nielsen et al, Science,1991, 254, 1497-1500.

PNA has been modified to incorporate numerous modifications since thebasic PNA structure was first prepared. The basic structure is shownbelow:

wherein

Bx is a heterocyclic base moiety;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the ω-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. A suitable class of linking groups have been selected togive a non-ionic oligomeric compound. The non-ionic morpholino-basedoligomeric compounds are less likely to have undesired interactions withcellular proteins. Morpholino-based oligomeric compounds are non-ionicmimics of oligonucleotides which are less likely to form undesiredinteractions with cellular proteins (Dwaine A. Braasch and David R.Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-basedoligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issuedJul. 23, 1991. The morpholino class of oligomeric compounds have beenprepared having a variety of different linking groups joining themonomeric subunits.

Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

wherein

T₁ is hydroxyl or a protected hydroxyl;

T₅ is hydrogen or a phosphate or phosphate derivative;

L₂ is a linking group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic is referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in an DNA/RNAmolecule is replaced with a cyclohenyl ring. CeNA DMT protectedphosphoramidite monomers have been prepared and used for oligomericcompound synthesis following classical phosphoramidite chemistry. Fullymodified CeNA oligomeric compounds and oligonucleotides having specificpositions modified with CeNA have been prepared and studied (see Wang etal., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general theincorporation of CeNA monomers into a DNA chain increases its stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Thestudy of incorporating CeNA structures into natural nucleic acidstructures was shown by NMR and circular dichroism to proceed with easyconformational adaptation. Furthermore the incorporation of CeNA into asequence targeting RNA was stable to serum and able to activate E. ColiRNase resulting in cleavage of the target RNA strand.

The general formula of CeNA is shown below:

wherein

each Bx is a heterocyclic base moiety;

T₁ is hydroxyl or a protected hydroxyl;

T₂ is hydroxyl or a protected hydroxyl;

L₃ is a linking group; and

n is from 2 to about 450.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid)can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

Another group of modifications includes nucleosides having sugarmoieties that are bicyclic thereby locking the sugar conformationalgeometry. The most studied of these nucleosides is a bicyclic sugarmoiety having a 4′-CH₂—O-2′ bridge. As can be seen in the structurebelow the 2′-O— has been linked via a methylene group to the 4′ carbon.This bridge attaches under the sugar as shown forcing the sugar ringinto a locked 3′-endo conformation geometry. The ∀-L nucleoside has alsobeen reported wherein the linkage is above the ring and the heterocyclicbase is in the ∀ rather than the ∃-conformation (see U.S. PatentApplication Publication No.: Application 2003/0087230). The xylo analoghas also been prepared (see U.S. Patent Application Publication No.:2003/0082807). The preferred bridge for a locked nucleic acid (LNA) is4′—(—CH₂—)_(n)—O-2′ wherein n is 1 or 2. The literature is confusingwhen the term locked nucleic acid is used but in general locked nucleicacids refers to n=1, ENA™ refers to n=2 (Kaneko et al., U.S. PatentApplication Publication No.: US 2002/0147332, Singh et al., Chem.Commun., 1998, 4, 455-456, also see U.S. Pat. Nos. 6,268,490 and6,670,461 and U.S. Patent Application Publication No.: US 2003/0207841).However the term locked nucleic acids can also be used in a more generalsense to describe any bicyclic sugar moiety that has a lockedconformation.

ENA™ along with LNA (n=1) have been studied more than the myriad ofother analogs. Oligomeric compounds incorporating LNA and ENA analogsdisplay very high duplex thermal stabilities with complementary DNA andRNA (Tm=+3 to +10 C), stability towards 3′-exonucleolytic degradationand good solubility properties.

The basic structure of LNA showing the bicyclic ring system is shownbelow:

wherein

each Bx is a heterocyclic base moiety;

each L₁ is an internucleoside linking group;

T₁ is hydroxyl or a protected hydroxyl;

T₂ is hydroxyl or a protected hydroxyl, and

n is from 1 to about 80.

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638.) The authors have demonstrated that LNAs confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. LIPOFECTIN™-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Some oligonucleotide mimetics have been prepared to include bicyclic andtricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J.Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogshave been oligomerized using the phosphoramidite approach and theresulting oligomeric compounds containing tricyclic nucleoside analogshave shown increased thermal stabilities (Tm's) when hybridized to DNA,RNA and itself. Oligomeric compounds containing bicyclic nucleosideanalogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acid and incorporates a phosphorus group inthe backbone. This class of olignucleotide mimetic is reported to haveuseful physical and biological and pharmacological properties in theareas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted sugar moieties. These oligomeric compounds comprise a sugarsubstituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable areO((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from1 to about 10. Some oligonucleotides comprise a sugar substituent groupselected from: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Onemodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. One modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other sugar substituent groups include methoxy (—O—CH₃), aminopropoxy(—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) andfluoro (F). 2′-Sugar substituent groups may be in the arabino (up)position or ribo (down) position. One 2′-arabino modification is 2′-F.Similar modifications may also be made at other positions on theoligomeric compound, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligomeric compounds may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Representative sugar substituent groups include groups of formula I_(a)or II_(a):

wherein:

R_(b) is O, S or NH;

R_(d) is a single bond, O, S or C(═O);

R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(m)),N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

R_(r) is —R_(x)—R_(y);

each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_(u) and R_(v), together form a phthalimido moiety withthe nitrogen atom to which they are attached;

each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

R_(x) is a bond or a linking moiety;

R_(y) is a chemical functional group, a conjugate group or a solidsupport medium;

each R_(m) and R_(n) is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein thesubstituent groups are selected from hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl where said acyl isan acid amide or an ester;

or R_(m) and R_(n), together, are a nitrogen protecting group, arejoined in a ring structure that optionally includes an additionalheteroatom selected from N and O or are a chemical functional group;

R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

R_(f), R_(g) and R_(h) comprise a ring system having from about 4 toabout 7 carbon atoms or having from about 3 to about 6 carbon atoms and1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m))OR_(k),halo, SR_(k) or CN;

m_(a) is 1 to about 10;

each mb is, independently, 0 or 1;

mc is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups are disclosed in U.S. patentapplication Ser. No. 09/130,973, filed Aug. 7, 1998, entitled “Capped2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in itsentirety.

Representative cyclic substituent groups are disclosed in U.S. patentapplication Ser. No. 09/123,108, filed Jul. 27, 1998, entitled “RNATargeted 2′-Oligomeric compounds that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Particular sugar substituent groups include O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃))₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups are disclosed in U.S. patentapplication Ser. No. 09/349,040, entitled “Functionalized Oligomers,”filed Jul. 7, 1999, hereby incorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Synthesis of Chimeric Oligonucleotides:

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3 “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers.”

(2′-O-Me)-(2′-deoxy)-(2′-O-Me) Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

(2′-O-(2-Methoxyethyl))-(2′-deoxy)-(2′-O-(Methoxyethyl)) ChimericPhosphorothioate Oligonucleotides

(2′-O-(2-methoxyethyl))-(2′-deoxy)-(−2′-O-(methoxyethyl)) chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

(2′-O-(2-Methoxyethyl)Phosphodiester)-(2′-deoxyPhosphorothioate)-(2′-O-(2-Methoxyethyl) Phosphodiester) ChimericOligonucleotides

(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxyphosphorothioate)-(2′-O-(methoxyethyl) phosphodiester) chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1, 1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as, but notlimited to, antisense mechanisms, including RNase H-mediated and RNAinterference mechanisms, as these mechanisms involved the hybridizationof a synthetic sequence strand to an RNA target strand. In the case ofRNase H, effective inhibition of the mRNA requires that the antisensesequence achieve at least a threshold of hybridization.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependent on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is alsocorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as illustrated in FIG. 2, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation.

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA-like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry (see Scheme 1). There is an apparent preferencefor an RNA type duplex (A form helix, predominantly 3′-endo) as arequirement (e.g. trigger) of RNA interference which is supported inpart by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosidesappears efficient in triggering RNAi response in the C. elegans system.Properties that are enhanced by using more stable 3′-endo nucleosidesinclude but aren't limited to modulation of pharmacokinetic propertiesthrough modification of protein binding, protein off-rate, absorptionand clearance; modulation of nuclease stability as well as chemicalstability; modulation of the binding affinity and specificity of theoligomer (affinity and specificity for enzymes as well as forcomplementary sequences); and increasing efficacy of RNA cleavage. Thepresent invention provides oligomeric compounds designed to act astriggers of RNAi having one or more nucleosides modified in such a wayas to favor a C3′-endo type conformation.

Along similar lines, oligomeric triggers of RNAi response might becomposed of one or more nucleosides modified in such a way thatconformation is locked into a C3′-endo type conformation, i.e. LockedNucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), andethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modifiednucleosides amenable to the present invention are shown below. Theseexamples are meant to be representative and not exhaustive.

Oligomeric compounds may also include nucleobase (often referred to inthe art simply as “base” or “heterocyclic base moiety”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesalso referred herein as heterocyclic base moieties include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Somenucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

In one aspect of the present invention oligomeric compounds are preparedhaving polycyclic heterocyclic compounds in place of one or moreheterocyclic base moieties. A number of tricyclic heterocyclic compoundshave been previously reported. These compounds are routinely used inantisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs. Many of these polycyclic heterocyclic compounds have thegeneral formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=H) (Kurchavov, et al, Nucleosides and Nucleotides, 1997, 16,1837-1846), 1,3-diazaphenothiazine-2-one (R₁₀=S, R₁₁-R₁₄=H), (Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388). When incorporated into oligonucleotides, these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.Patent Application Publication 20030207804 and U.S. Patent ApplicationPublication 20030175906, both of which are incorporated herein byreference in their entirety).

Helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀=O, R₁₁=—O—(CH₂)₂—NH₂, R₁₂₋₁₄=H)(Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532).Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification. On the other hand, the gain in helical stabilitydoes not compromise the specificity of the oligonucleotides. The T_(m)data indicate an even greater discrimination between the perfect matchand mismatched sequences compared to dC5^(me). It was suggested that thetethered amino group serves as an additional hydrogen bond donor tointeract with the Hoogsteen face, namely the O6, of a complementaryguanine thereby forming 4 hydrogen bonds. This means that the increasedaffinity of G-clamp is mediated by the combination of extended basestacking and additional specific hydrogen bonding.

Tricyclic heterocyclic compounds and methods of using them that areamenable to the present invention are disclosed in U.S. Pat. Nos.6,028,183, and 6,007,992, the contents of both are incorporated hereinin their entirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their sequence specificity makes them valuable nucleobase analogsfor the development of more potent antisense-based drugs. In fact,promising data have been derived from in vitro experiments demonstratingthat heptanucleotides containing phenoxazine substitutions can activateRNaseH, enhance cellular uptake and exhibit an increased antisenseactivity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120,8531-8532). The activity enhancement was even more pronounced in case ofG-clamp, as a single substitution was shown to significantly improve thein vitro potency of a 20mer 2′-deoxyphosphorothioate oligonucleotides(Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner,R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).

Modified polycyclic heterocyclic compounds useful as heterocyclic basesare disclosed in but not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653;5,763,588; 6,005,096; and 5,681,941, and U.S. Patent ApplicationPublication 20030158403, each of which is incorporated herein byreference in its entirety.

One substitution that can be appended to the oligomeric compounds of theinvention involves the linkage of one or more moieties or conjugateswhich enhance the activity, cellular distribution or cellular uptake ofthe resulting oligomeric compounds. In one embodiment such modifiedoligomeric compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, carbohydrates, lipids, phospholipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve oligomer uptake, enhance oligomer resistance todegradation, and/or strengthen hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Representative conjugate groups are disclosedin International Patent Application PCT/US92/09196, filed Oct. 23, 1992the entire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of oligomeric compounds toenhance properties such as for example nuclease stability. Included instabilizing groups are cap structures. By “cap structure or terminal capmoiety” is meant chemical modifications, which have been incorporated ateither terminus of oligonucleotides (see for example Wincott et al., WO97/26270, incorporated by reference herein). These terminalmodifications protect the oligomeric compounds having terminal nucleicacid molecules from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present onboth termini. For double-stranded oligomeric compounds, the cap may bepresent at either or both termini of either strand. In non-limitingexamples, the 5′-cap includes inverted abasic residue (moiety),4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl riucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety (see Wincott et al., International PCTpublication No. WO 97/26270, incorporated by reference herein).

Particularly preferred 3′-cap structures of the present inventioninclude, for example 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602 published on Jan. 16, 2003.

It is not necessary for all positions in an oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within aoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds that contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, an oligomeric compound may be designed tocomprise a region that serves as a substrate for RNase H. RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H by an oligomeric compound having a cleavageregion, therefore, results in cleavage of the RNA target, therebyenhancing the efficiency of the oligomeric compound. Consequently,comparable results can often be obtained with shorter oligomericcompounds having substrate regions when chimeras are used, compared tofor example phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotidemimics, oligonucleotide analogs, oligonucleosides and/or oligonucleotidemimetics as described above. Such oligomeric compounds have also beenreferred to in the art as hybrids, hemimers, gapmers or invertedgapmers. Representative U.S. patents that teach the preparation of suchhybrid structures include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference in its entirety.

The conformation of modified nucleosides and their oligomers can beestimated by various methods such as molecular dynamics calculations,nuclear magnetic resonance spectroscopy and CD measurements. Hence,modifications predicted to induce RNA-like conformations (A-form duplexgeometry in an oligomeric context), are useful in the oligomericcompounds of the present invention. The synthesis of modifiednucleosides amenable to the present invention are known in the art (seefor example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. LeroyB. Townsend, 1988, Plenum Press.)

In one aspect, the present invention is directed to oligomeric compoundsthat are designed to have enhanced properties compared to native RNA.One method to design optimized or enhanced oligomeric compounds involveseach nucleoside of the selected sequence being scrutinized for possibleenhancing modifications. One modification would be the replacement ofone or more RNA nucleosides with nucleosides that have the same 3′-endoconformational geometry. Such modifications can enhance chemical andnuclease stability relative to native RNA while at the same time beingmuch cheaper and easier to synthesize and/or incorporate into anoligonucleotide. The sequence can be further divided into regions andthe nucleosides of each region evaluated for enhancing modificationsthat can be the result of a chimeric configuration. Consideration isalso given to the 5′ and 3′-termini as there are often advantageousmodifications that can be made to one or more of the terminalnucleosides. The oligomeric compounds of the present invention mayinclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double-stranded sequence or sequences.Other modifications considered are internucleoside linkages, conjugategroups, substitute sugars or bases, substitution of one or morenucleosides with nucleoside mimetics and any other modification that canenhance the desired property of the oligomeric compound.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituentalso have been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotideshaving the 2′-MOE modification displayed improved RNA affinity andhigher nuclease resistance. Chimeric oligonucleotides having 2′-MOEsubstituents in the wing nucleosides and an internal region ofdeoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotideor gapmer) have shown effective reduction in the growth of tumors inanimal models at low doses. 2′-MOE substituted oligonucleotides havealso shown outstanding promise as antisense agents in several diseasestates. One such MOE substituted oligonucleotide is presently beinginvestigated in clinical trials for the treatment of CMV retinitis.

Unless otherwise defined herein, alkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆,straight or (where possible) branched chain aliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, C₁-C₈, orC₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbylcontaining at least one, or about 1 to about 3 hetero atoms in thechain, including the terminal portion of the chain. Suitable heteroatomsinclude N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, C₃-C₈, orC₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkenyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkynyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon. Thenumber of carbon atoms can vary from 1 to about 12, from 1 to about 6,and the total number of ring members varies from three to about 15, orfrom about 3 to about 8. Suitable ring heteroatoms are N, O and S.Suitable heterocycloalkyl groups include, but are not limited to,morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl,homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl,tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl,tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, andtetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Suitable aryl rings haveabout 6 to about 20 ring carbons. Especially suitable aryl rings includephenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. The ring system can contain about 1 to about 4 rings.The number of carbon atoms can vary from 1 to about 12, from 1 to about6, and the total number of ring members varies from three to about 15,or from about 3 to about 8. Suitable ring heteroatoms are N, O and S.Suitable hetaryl moieties include, but are not limited to, pyrazolyl,thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl,purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl,etc.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings. Suitable halo (halogen) substituents are Cl, Br, andI.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc.

In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate.

Phosphate protecting groups include those described in U.S. Pat. Nos.5,760,209, 5,614,621, 6,051,699, 6,020,475, 6,326,478, 6,169,177,6,121,437, 6,465,628 each of which is expressly incorporated herein byreference in its entirety.

Screening methods for the identification of effective modulators ofsmall non-coding RNAs are also comprehended by the instant invention andcomprise the steps of contacting a small non-coding RNA, or portionthereof, with one or more candidate modulators, and selecting for one ormore candidate modulators which decrease or increase the levels,expression or alter the function of the small non-coding RNA. Once it isshown that the candidate modulator or modulators are capable ofmodulating (e.g. either decreasing or increasing) the levels, expressionor altering the function of the small non-coding RNA, the modulator maythen be employed in further investigative studies, or for use as atarget validation, research, diagnostic, or therapeutic agent inaccordance with the present invention.

Screening methods for the identification of small non-coding RNA mimicsare also within the scope of the invention. Screening for smallnon-coding RNA modulators or mimics can also be performed in vitro, exvivo, or in vivo by contacting samples, tissues, cells or organisms withcandidate modulators or mimics and selecting for one or more candidatemodulators which show modulatory effects.

Design and Screening of Duplexed Oligomeric Compounds:

In screening and target validation studies, oligomeric compounds of theinvention can be used in combination with their respective complementarystrand oligomeric compound to form stabilized double-stranded (duplexed)oligonucleotides. In accordance with the present invention, a series ofduplexes comprising the oligomeric compounds of the present inventionand their complements can be designed to target a small non-coding RNA.The ends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in some embodiments, both strands of theduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini, as described supra.

In some embodiments, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181) may be prepared with bluntends (no single stranded overhang) as shown:

In other embodiments, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG, having a two-nucleobase overhang ofdeoxythymidine (dT) and its complement sense strand may be prepared withoverhangs as shown:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.).

For use in drug discovery, oligomeric compounds of the present inventionare used to elucidate relationships that exist between small non-codingRNAs, genes or proteins and a disease state, phenotype, or condition.These methods include detecting or modulating a target comprisingcontacting a sample, tissue, cell, or organism with the oligomericcompounds and compositions of the present invention, measuring thelevels of the target and/or the levels of downstream gene productsincluding mRNA or proteins encoded thereby, a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to an untreated sample, a positive control or anegative control. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa disease.

The oligomeric compounds and compositions of the present invention canadditionally be utilized for diagnostics, therapeutics, prophylaxis andas research reagents and kits. Such uses allows for those of ordinaryskill to elucidate the function of particular non-coding or codingnucleic acids or to distinguish between functions of various members ofa biological pathway.

For use in kits and diagnostics, the oligomeric compounds andcompositions of the present invention, either alone or in combinationwith other compounds or therapeutics, can be used as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of non-coding or codingnucleic acids expressed within cells and tissues.

As one non-limiting example, expression patterns within cells or tissuestreated with one or more oligomeric compounds or compositions of theinvention are compared to control cells or tissues not treated with thecompounds or compositions and the patterns produced are analyzed fordifferential levels of nucleic acid expression as they pertain, forexample, to disease association, signaling pathway, cellularlocalization, expression level, size, structure or function of the genesexamined. These analyses can be performed on stimulated or unstimulatedcells and in the presence or absence of other compounds that affectexpression patterns.

Cell Culture and Oligonucleotide Treatment:

The effects of oligomeric compounds on target nucleic acid expression orfunction can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can bereadily determined by methods routine in the art, for example Northernblot analysis, ribonuclease protection assays, or real-time RT-PCR. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is present inthe cell type chosen.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 is obtainedfrom the American Type Culture Collection (ATCC) (Manassas, Va.). T-24cells were routinely cultured in complete McCoy's 5A basal media(Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetalcalf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. ForNorthern blotting or other analyses, cells harvested when they reached90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria#353872) at a density of 7000 cells/well for use in real-time RT-PCRanalysis.

A549 Cells:

The human lung carcinoma cell line A549 is obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

HMECs:

Normal human mammary epithelial cells (HMECs) are obtained from AmericanType Culture Collection (Manassus, Va.). HMECs are routinely cultured inDMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.). Cells are routinely passaged by trypsinization anddilution when they reach approximately 90% confluence. HMECs are platedin 24-well plates (Falcon-Primaria # 353047, BD Biosciences, Bedford,Mass.) at a density of 50,000-60,000 cells per well, and allowed toattach overnight prior to treatment with oligomeric compounds. HMECs areplated in 96-well plates (Falcon-Primaria #353872, BD Biosciences,Bedford, Mass.) at a density of approximately 10,000 cells per well andallowed to attach overnight prior to treatment with oligomericcompounds.

MCF7 Cells:

The breast carcinoma cell line MCF7 is obtained from American TypeCulture Collection (Manassus, Va.). MCF7 cells are routinely cultured inDMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.). Cells are routinely passaged by trypsinization anddilution when they reach approximately 90% confluence. MCF7 cells areplated in 24-well plates (Falcon-Primaria # 353047, BD Biosciences,Bedford, Mass.) at a density of approximately 140,000 cells per well,and allowed to attach overnight prior to treatment with oligomericcompounds. MCF7 cells are plated in 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately20,000 cells per well and allowed to attach overnight prior to treatmentwith oligomeric compounds.

T47D Cells:

The breast carcinoma cell line T47D is obtained from American TypeCulture Collection (Manassus, Va.). T47D cells are deficient inexpression of the tumor suppressor gene p53. T47D cells are cultured inDMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.). Cells are routinely passaged by trypsinization anddilution when they reach approximately 90% confluence. T47D cells areplated in 24-well plates (Falcon-Primaria #353047, BD Biosciences,Bedford, Mass.) at a density of approximately 170,000 cells per well,and allowed to attach overnight prior to treatment with oligomericcompounds. T47D cells are plated in 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately20,000 cells per well and allowed to attach overnight prior to treatmentwith oligomeric compounds.

BJ Cells:

The normal human foreskin fibroblast BJ cell line was obtained fromAmerican Type Culture Collection (Manassas, Va.). BJ cells wereroutinely cultured in MEM high glucose with 2 mM L-glutamine and Earle'sBSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mMsodium pyruvate (all media and supplements from Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 80%confluence. Cells were plated on collagen-coated 24-well plates(Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately50,000 cells per well, and allowed to attach to wells overnight.

B16-F10 Cells:

The mouse melanoma cell line B16-F10 was obtained from American TypeCulture Collection (Manassas, Va.). B16-F10 cells were routinelycultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum (Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 80%confluence. Cells were seeded into collagen-coated 24-well plates(Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately50,000 cells per well and allowed to attach overnight.

HUVECs:

Human vascular endothelial cells (HUVECs) are obtained from AmericanType Culture Collection (Manassus, Va.). HUVECs are routinely culturedin EBM (Clonetics Corporation, Walkersville, Md.) supplemented withSingleQuots supplements (Clonetics Corporation, Walkersville, Md.).Cells are routinely passaged by trypsinization and dilution when theyreach approximately 90% confluence and are maintained for up to 15passages. HUVECs are plated at approximately 3000 cells/well in 96-wellplates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) andtreated with oligomeric compounds one day later.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) cells are obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) are obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

293T Cells:

The human 293T cell line is obtained from American Type CultureCollection (Manassas, Va.). 293T cells are a highly transfectable cellline constitutively expressing the simian virus 40 (SV40) large Tantigen. 293T cells were maintained in Dulbeccos' Modified Medium (DMEM)(Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetalcalf serum and antibiotics (Life Technologies).

HepG2 Cells:

The human hepatoblastoma cell line HepG2 is obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). HepG2 cells areroutinely cultured in Eagle's MEM supplemented with 10% fetal bovineserum, 1 mM non-essential amino acids, and 1 mM sodium pyruvate (mediumand all supplements from Invitrogen Life Technologies, Carlsbad,Calif.). Cells are routinely passaged by trypsinization and dilutionwhen they reach approximately 90% confluence. For treatment witholigomeric compounds, cells are seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof approximately 7000 cells/well prior to treatment with oligomericcompounds. For the caspase assay, cells are seeded into collagen coated96-well plates (BIOCOAT cellware, Collagen type I, B-D #354407/356407,Becton Dickinson, Bedford, Mass.) at a density of 7500 cells/well.

Preadipocytes:

Human preadipocytes are obtained from Zen-Bio, Inc. (Research TrianglePark, N.C.). Preadipocytes were routinely maintained in PreadipocyteMedium (ZenBio, Inc., Research Triangle Park, N.C.) supplemented withantibiotics as recommended by the supplier. Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were routinely maintained for up to 5 passages asrecommended by the supplier. To induce differentiation of preadipocytes,cells are then incubated with differentiation media consisting ofPreadipocyte Medium further supplemented with 2% more fetal bovine serum(final total of 12%), amino acids, 100 nM insulin, 0.5 mM IBMX, 1 μMdexamethasone and 1 μM BRL49653. Cells are left in differentiation mediafor 3-5 days and then re-fed with adipocyte media consisting ofPreadipocyte Medium supplemented with 33 μM biotin, 17 μM pantothenate,100 nM insulin and 1 μM dexamethasone. Cells differentiate within oneweek. At this point cells are ready for treatment with the oligomericcompounds of the invention. One day prior to transfection, 96-wellplates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) areseeded with approximately 3000 cells/well prior to treatment witholigomeric compounds.

Differentiated Adipocytes:

Human adipocytes are obtained from Zen-Bio, Inc. (Research TrianglePark, N.C.). Adipocytes were routinely maintained in Adipocyte Medium(ZenBio, Inc., Research Triangle Park, N.C.) supplemented withantibiotics as recommended by the supplier. Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were routinely maintained for up to 5 passages asrecommended by the supplier.

NT2 Cells:

The NT2 cell line is obtained from the American Type Culture Collection(ATCC; Manassas, Va.). The NT2 cell line, which has the ATCC designationNTERA-2 c1.D1, is a pluripotent human testicular embryonal carcinomacell line derived by cloning the NTERA-2 cell line. The parental NTERA-2line was established in 1980 from a nude mouse xenograft of the Tera-2cell line (ATCC HTB-106). NT2 cells were routinely cultured in DMEM,high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplementedwith 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.).Cells were routinely passaged by trypsinization and dilution when theyreached 90% confluence. For Northern blotting or other analyses, cellsharvested when they reached 90% confluence.

HeLa Cells:

The human epitheloid carcinoma cell line HeLa is obtained from theAmerican Tissue Type Culture Collection (Manassas, Va.). HeLa cells wereroutinely cultured in DMEM, high glucose (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. ForNorthern blotting or other analyses, cells were harvested when theyreached 90% confluence.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Oligomeric Compounds:

In general, when cells reach approximately 80% confluency, they aretreated with oligomeric compounds of the invention. Oligomeric compoundsare introduced into cells using the cationic lipid transfection reagentLIPOFECTIN™ (Invitrogen Life Technologies, Carlsbad, Calif.). Oligomericcompounds are mixed with LIPOFECTIN™ in OPTI-MEM™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired finalconcentration of oligomeric compound and LIPOFECTIN™. Before adding tocells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ are mixedthoroughly and incubated for approximately 0.5 hrs. The medium isremoved from the plates and the plates are tapped on sterile gauze. Eachwell of a 96-well plate is washed with 150 μl of phosphate-bufferedsaline or Hank's balanced salt solution. Each well of a 24-well plate iswashed with 250 μL of phosphate-buffered saline or Hank's balanced saltsolution. The wash buffer in each well is replaced with 100 μL or 250 μLof the oligomeric compound/OPTI-MEM™/LIPOFECTIN™ cocktail for 96-well or24-well plates, respectively. Untreated control cells receiveLIPOFECTIN™ only. The plates are incubated for approximately 4 to 7hours at 37° C., after which the medium is removed and the plates aretapped on sterile gauze. 100 μl or 1 mL of full growth medium is addedto each well of a 96-well plate or a 24-well plate, respectively. Cellsare harvested 16-24 hours after oligonucleotide treatment, at which timeRNA can be isolated and target reduction measured by real-time RT-PCR,or other phenotypic assays performed. In general, data from treatedcells are obtained in triplicate, and results presented as an average ofthe three trials.

In some embodiments, cells are transiently transfected with oligomericcompounds of the instant invention. In some embodiments, cells aretransfected and selected for stable expression of an oligomeric compoundof the instant invention.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide may be selected from ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2) or another suitable positivecontrol. Controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethylsshown in bold) with a phosphorothioate backbone or having chemicalmodifications similar to the oligonucleotides being tested. For mouse orrat cells the positive control oligonucleotide may be ISIS 15770(ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3), a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS15770) or other suitable control target RNA may then be utilized as thescreening concentration for new oligonucleotides in subsequentexperiments for that cell line. If 80% inhibition is not achieved, thelowest concentration of positive control oligonucleotide that results in60% inhibition of target expression or function is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. The concentrations of oligonucleotides used herein canrange from 10 nM to 300 nM.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904), mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41) andreal-time quantitative RT-PCR (Heid, et al., Genome Res., 1996, 6(10),986-94).

Analysis of Oligonucleotide Inhibition of a Target Levels or Expression:

Modulation of target levels or expression can be assayed in a variety ofways known in the art. For example, target nucleic acid levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time quantitative RT-PCR (also known asRT-PCR). Real-time quantitative RT-PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Real-time quantitative RT-PCR canbe conveniently accomplished using the commercially available ABI PRISM™7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

RNA Isolation:

Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al, (Clin. Chem., 1996,42, 1758-1764). Other methods for poly(A)+mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL coldphosphate-buffered saline (PBS). 60 μL lysis buffer (10 mM Tris-HCl, pH7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleosidecomplex) was added to each well, the plate was gently agitated and thenincubated at room temperature for five minutes. 55 μL of lysate wastransferred to Oligo d(T) coated 96-well plates (AGCT Inc., IrvineCalif.). Plates were incubated for 60 minutes at room temperature,washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mMEDTA, 0.3 M NaCl). After the final wash, the plate was blotted on papertowels to remove excess wash buffer and then air-dried for 5 minutes. 60μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., wasadded to each well, the plate was incubated on a 90° C. hot plate for 5minutes, and the eluate was then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Real-Time Quantitative PCR Analysis of a Target RNA Levels:

Quantitation of a target RNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of RNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer/probe sets specific to thetarget gene (or RNA) being measured are evaluated for their ability tobe “multiplexed” with a GAPDH amplification reaction. In multiplexing,both the target gene (or RNA) and the internal standard gene GAPDH areamplified concurrently in a single sample. In this analysis, RNAisolated from untreated cells is serially diluted. Each dilution isamplified in the presence of primer/probe sets specific for GAPDH only,target gene (or RNA) only (“single-plexing”), or both (multiplexing).Following PCR amplification, standard curves of GAPDH and target RNAsignal as a function of dilution are generated from both thesingle-plexed and multiplexed samples. If both the slope and correlationcoefficient of the GAPDH and target signals generated from themultiplexed samples fall within 10% of their corresponding valuesgenerated from the single-plexed samples, the primer/probe set specificfor that target is deemed multiplexable. Other methods of PCR are alsoknown in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene (or RNA) target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers are designed to hybridize to the target sequence.

Northern Blot Analysis of Target RNA Levels:

Eighteen hours after treatment, cell monolayers were washed twice withcold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood,Tex.). Total RNA was prepared following manufacturer's recommendedprotocols. Twenty micrograms of total RNA was fractionated byelectrophoresis through 1.2% agarose gels containing 1.1% formaldehydeusing a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA wastransferred from the gel to HYBOND™-N+nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect a target, a target specific primer/probe set is prepared foranalysis by PCR. To normalize for variations in loading and transferefficiency, membranes can be stripped and probed for humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, PaloAlto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data can be normalized to GAPDH levels in untreatedcontrols.

The compounds and compositions of the invention are useful for researchand diagnostics, because these compounds and compositions hybridize tonucleic acids or interfere with the normal function of these nucleicacids. Hybridization of the compounds and compositions of the inventionwith a nucleic acid can be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the compound orcomposition, radiolabeling or any other suitable detection means. Kitsusing such detection means for detecting the level of selected proteinsin a sample may also be prepared.

The specificity and sensitivity of compounds and compositions can alsobe harnessed by those of skill in the art for therapeutic uses.Antisense oligomeric compounds have been employed as therapeuticmoieties in the treatment of disease states in animals, includinghumans. Antisense oligonucleotide drugs, including ribozymes, have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that oligomericcompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder presenting conditions that can be treated,ameliorated, or improved by modulating the expression of a selectedsmall non-coding target nucleic acid is treated by administering thecompounds and compositions. For example, in one non-limiting embodiment,the methods comprise the step of administering to or contacting theanimal, an effective amount of a modulator or mimic to treat, ameliorateor improve the conditions associated with the disease or disorder. Thecompounds of the present invention effectively modulate the activity orfunction of the small non-coding RNA target or inhibit the expression orlevels of the small non-coding RNA target. In one embodiment, theactivity or expression of the target in an animal is inhibited by about10%. In another embodiment the activity or expression of a target in ananimal is inhibited by about 30%. Further, the activity or expression ofa target in an animal is inhibited by 50% or more, by 60% or more, by70% or more, by 80% or more, by 90% or more, or by 95% or more. Inanother embodiment, the present invention provides for the use of acompound of the invention in the manufacture of a medicament for thetreatment of any and all conditions disclosed herein.

The reduction of target levels may be measured in serum, adipose tissue,liver or any other body fluid, tissue or organ of the animal known tocontain the small non-coding RNA or its precursor. Further, the cellscontained within the fluids, tissues or organs being analyzed contain anucleic acid molecule of a downstream target regulated or modulated bythe small non-coding RNA target itself.

The oligomeric compounds and compositions of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofthe compound or composition to a suitable pharmaceutically acceptablediluent or carrier. Use of the oligomeric compounds and methods of theinvention may also be useful prophylactically.

The oligomeric compounds and compositions of the invention may also beadmixed, encapsulated, conjugated or otherwise associated with othermolecules, molecule structures or mixtures of compounds, as for example,liposomes, receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative U.S. patents that teach the preparation of such uptake,distribution and/or absorption-assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The oligomeric compounds and compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the oligomeric compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligomeric compounds of the invention can be prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al, published Dec. 9, 1993 or inWO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al Largeroligomeric compounds that are processed to supply, as cleavage products,compounds capable of modulating the function or expression of smallnon-coding RNAs or their downstream targets are also consideredprodrugs.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds and compositionsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. Suitable examples include, but are notlimited to, sodium and potassium salts. For oligonucleotides, examplesof pharmaceutically acceptable salts and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

The present invention also includes pharmaceutical compositions andformulations that include the oligomeric compounds and compositions ofthe invention. The pharmaceutical compositions of the present inventionmay be administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

Oligomeric compounds may be formulated for delivery in vivo in anacceptable dosage form, e.g. as parenteral or non-parenteralformulations. Parenteral formulations include intravenous (IV),subcutaneous (SC), intraperitoneal (IP), intravitreal and intramuscular(IM) formulations, as well as formulations for delivery via pulmonaryinhalation, intranasal administration, topical administration, etc.Non-parenteral formulations include formulations for delivery via thealimentary canal, e.g. oral administration, rectal administration,intrajejunal instillation, etc. Rectal administration includesadministration as an enema or a suppository. Oral administrationincludes administration as a capsule, a gel capsule, a pill, an elixir,etc.

In some embodiments, an oligomeric compound can be administered to asubject via an oral route of administration. The subject may be ananimal or a human (man). An animal subject may be a mammal, such as amouse, a rat, a dog, a guinea pig, a monkey, a non-human primate, a cator a pig. Non-human primates include monkeys and chimpanzees. A suitableanimal subject may be an experimental animal, such as a mouse, rat,mouse, a rat, a dog, a monkey, a non-human primate, a cat or a pig.

In some embodiments, the subject may be a human. In certain embodiments,the subject may be a human patient. In certain embodiments, the subjectmay be in need of modulation of expression of one or more genes asdiscussed in more detail herein. In some particular embodiments, thesubject may be in need of inhibition of expression of one or more genesas discussed in more detail herein. In particular embodiments, thesubject may be in need of modulation, i.e. inhibition or enhancement, ofa nucleic acid target in order to obtain therapeutic indicationsdiscussed in more detail herein.

In some embodiments, non-parenteral (e.g. oral) oligomeric compoundformulations according to the present invention result in enhancedbioavailability of the compound. In this context, the term“bioavailability” refers to a measurement of that portion of anadministered drug which reaches the circulatory system (e.g. blood,especially blood plasma) when a particular mode of administration isused to deliver the drug. Enhanced bioavailability refers to aparticular mode of administration's ability to deliver oligonucleotideto the peripheral blood plasma of a subject relative to another mode ofadministration. For example, when a non-parenteral mode ofadministration (e.g. an oral mode) is used to introduce the drug into asubject, the bioavailability for that mode of administration may becompared to a different mode of administration, e.g. an IV mode ofadministration. In some embodiments, the area under a compound's bloodplasma concentration curve (AUC₀) after non-parenteral (e.g. oral,rectal, intrajejunal) administration may be divided by the area underthe drug's plasma concentration curve after intravenous (i.v.)administration (AUC_(iv)) to provide a dimensionless quotient (relativebioavailability, RB) that represents the fraction of compound absorbedvia the non-parenteral route as compared to the IV route. Acomposition's bioavailability is said to be enhanced in comparison toanother composition's bioavailability when the first composition'srelative bioavailability (RB₁) is greater than the second composition'srelative bioavailability (RB₂).

In general, bioavailability correlates with therapeutic efficacy when acompound's therapeutic efficacy is related to the blood concentrationachieved, even if the drug's ultimate site of action is intracellular(van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300).Bioavailability studies have been used to determine the degree ofintestinal absorption of a drug by measuring the change in peripheralblood levels of the drug after an oral dose (DiSanto, Chapter 76 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 1451-1458).

In general, an oral composition's bioavailability is said to be“enhanced” when its relative bioavailability is greater than thebioavailability of a composition substantially consisting of pureoligonucleotide, i.e. oligonucleotide in the absence of a penetrationenhancer.

Organ bioavailability refers to the concentration of compound in anorgan. Organ bioavailability may be measured in test subjects by anumber of means, such as by whole-body radiography. Organbioavailability may be modified, e.g. enhanced, by one or moremodifications to the oligomeric compound, by use of one or more carriercompounds or excipients. In general, an increase in bioavailability willresult in an increase in organ bioavailability.

Oral oligomeric compound compositions according to the present inventionmay comprise one or more “mucosal penetration enhancers,” also known as“absorption enhancers” or simply as “penetration enhancers.”Accordingly, some embodiments of the invention comprise at least oneoligomeric compound in combination with at least one penetrationenhancer. In general, a penetration enhancer is a substance thatfacilitates the transport of a drug across mucous membrane(s) associatedwith the desired mode of administration, e.g. intestinal epithelialmembranes. Accordingly it is desirable to select one or more penetrationenhancers that facilitate the uptake of one or more oligomericcompounds, without interfering with the activity of the compounds, andin such a manner the compounds can be introduced into the body of ananimal without unacceptable side-effects such as toxicity, irritation orallergic response.

Embodiments of the present invention provide compositions comprising oneor more pharmaceutically acceptable penetration enhancers, and methodsof using such compositions, which result in the improved bioavailabilityof oligomeric compounds administered via non-parenteral modes ofadministration. Heretofore, certain penetration enhancers have been usedto improve the bioavailability of certain drugs. See Muranishi, Crit.Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev.Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that theuptake and delivery of oligonucleotides can be greatly improved evenwhen administered by non-parenteral means through the use of a number ofdifferent classes of penetration enhancers.

In some embodiments, compositions for non-parenteral administrationinclude one or more modifications from naturally-occurringoligonucleotides (i.e. full-phosphodiester deoxyribosyl orfull-phosphodiester ribosyl oligonucleotides). Such modifications mayincrease binding affinity, nuclease stability, cell or tissuepermeability, tissue distribution, or other biological orpharmacokinetic property. Modifications may be made to the base, thelinker, or the sugar, in general, as discussed in more detail hereinwith regards to oligonucleotide chemistry. In some embodiments of theinvention, compositions for administration to a subject, and inparticular oral compositions for administration to an animal or humansubject, will comprise modified oligonucleotides having one or moremodifications for enhancing affinity, stability, tissue distribution, orother biological property.

Suitable modified linkers include phosphorothioate linkers. In someembodiments according to the invention, the oligomeric compound has atleast one phosphorothioate linker. Phosphorothioate linkers providenuclease stability as well as plasma protein binding characteristics tothe compound. Nuclease stability is useful for increasing the in vivolifetime of oligomeric compounds, while plasma protein binding decreasesthe rate of first pass clearance of oligomeric compound via renalexcretion. In some embodiments according to the present invention, theoligomeric compound has at least two phosphorothioate linkers. In someembodiments, wherein the oligomeric compound has exactly n nucleosides,the oligomeric compound has from one to n−1 phosphorothioate linkages.In some embodiments, wherein the oligomeric compound has exactly nnucleosides, the oligomeric compound has n−1 phosphorothioate linkages.In other embodiments wherein the oligomeric compound has exactly nnucleoside, and n is even, the oligomeric compound has from 1 to n/2phosphorothioate linkages, or, when n is odd, from 1 to (n−1)/2phosphorothioate linkages. In some embodiments, the oligomeric compoundhas alternating phosphodiester (PO) and phosphorothioate (PS) linkages.In other embodiments, the oligomeric compound has at least one stretchof two or more consecutive PO linkages and at least one stretch of twoor more PS linkages. In other embodiments, the oligomeric compound hasat least two stretches of PO linkages interrupted by at least one PSlinkage.

In some embodiments, at least one of the nucleosides is modified on theribosyl sugar unit by a modification that imparts nuclease stability,binding affinity or some other beneficial biological property to thesugar. In some cases, the sugar modification includes a 2′-modification,e.g. the 2′-OH of the ribosyl sugar is replaced or substituted. Suitablereplacements for 2′-OH include 2′-F and 2′-arabino-F. Suitablesubstitutions for OH include 2′-O-alkyl, e.g. 2′-O-methyl, and2′-O-substituted alkyl, e.g. 2′-O-methoxyethyl, 2′-O-aminopropyl, etc.In some embodiments, the oligomeric compound contains at least one2′-modification. In some embodiments, the oligomeric compound containsat least 22′-modifications. In some embodiments, the oligomeric compoundhas at least one 2′-modification at each of the termini (i.e. the 3′-and 5′-terminal nucleosides each have the same or different2′-modifications). In some embodiments, the oligomeric compound has atleast two sequential 2′-modifications at each end of the compound. Insome embodiments, oligomeric compounds further comprise at least onedeoxynucleoside. In particular embodiments, oligomeric compoundscomprise a stretch of deoxynucleosides such that the stretch is capableof activating RNase (e.g. RNase H) cleavage of an RNA to which theoligomeric compound is capable of hybridizing. In some embodiments, astretch of deoxynucleosides capable of activating RNase-mediatedcleavage of RNA comprises about 8 to about 16, e.g. about 8 to about 16consecutive deoxynucleosides. In further embodiments, oligomericcompounds are capable of eliciting cleaveage by dsRNAse enzymes.

Oral compositions for administration of non-parenteral oligomericcompounds and compositions of the present invention may be formulated invarious dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. The term“alimentary delivery” encompasses e.g. oral, rectal, endoscopic andsublingual/buccal administration. A common requirement for these modesof administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of thenucleic acid(s) so administered.

Delivery of a drug via the oral mucosa, as in the case of buccal andsublingual administration, has several desirable features, including, inmany instances, a more rapid rise in plasma concentration of the drugthan via oral delivery (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711).

Endoscopy may be used for delivery directly to an interior portion ofthe alimentary tract. For example, endoscopic retrogradecystopancreatography (ERCP) takes advantage of extended gastroscopy andpermits selective access to the biliary tract and the pancreatic duct(Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591).Pharmaceutical compositions, including liposomal formulations, can bedelivered directly into portions of the alimentary canal, such as, e.g.,the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastricsubmucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) viaendoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs,1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington etal., Ailment Pharmacol Ther., 1995, 9, 471) can also be used for directalimentary delivery of pharmaceutical compositions.

In some embodiments, oligomeric compound formulations may beadministered through the anus into the rectum or lower intestine. Rectalsuppositories, retention enemas or rectal catheters can be used for thispurpose and may be preferred when patient compliance might otherwise bedifficult to achieve (e.g., in pediatric and geriatric applications, orwhen the patient is vomiting or unconscious). Rectal administration canresult in more prompt and higher blood levels than the oral route.(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Becauseabout 50% of the drug that is absorbed from the rectum will bypass theliver, administration by this route significantly reduces the potentialfor first-pass metabolism (Benet et al., Chapter 1 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996).

Some embodiments of the present invention employ various penetrationenhancers in order to effect transport of oligomeric compounds andcompositions across mucosal and epithelial membranes. Penetrationenhancers may be classified as belonging to one of five broadcategories—surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92). Penetration enhancersand their uses are described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Accordingly, some embodimentscomprise oral oligomeric compound compositions comprising at least onemember of the group consisting of surfactants, fatty acids, bile salts,chelating agents, and non-chelating surfactants. Further embodimentscomprise oral oligomeric compound comprising at least one fatty acid,e.g. capric or lauric acid, or combinations or salts thereof. Otherembodiments comprise methods of enhancing the oral bioavailability of anoligomeric compound, the method comprising co-administering theoligomeric compound and at least one penetration enhancer.

Other excipients that may be added to oral oligomeric compoundcompositions include surfactants (or “surface-active agents”), which arechemical entities which, when dissolved in an aqueous solution, reducethe surface tension of the solution or the interfacial tension betweenthe aqueous solution and another liquid, with the result that absorptionof oligomeric compounds through the alimentary mucosa and otherepithelial membranes is enhanced. In addition to bile salts and fattyacids, surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J.Pharm. Phamacol., 1988, 40, 252).

Fatty acids and their derivatives which act as penetration enhancers andmay be used in compositions of the present invention include, forexample, oleic acid, lauric acid, capric acid (n-decanoic acid),myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol),dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcamitines, acylcholines and mono- anddi-glycerides thereof and/or physiologically acceptable salts thereof(i.e., oleate, laurate, caprate, myristate, palmitate, stearate,linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

In some embodiments, oligomeric compound compositions for oral deliverycomprise at least two discrete phases, which phases may compriseparticles, capsules, gel-capsules, microspheres, etc. Each phase maycontain one or more oligomeric compounds, penetration enhancers,surfactants, bioadhesives, effervescent agents, or other adjuvant,excipient or diluent. In some embodiments, one phase comprises at leastone oligomeric compound and at least one penetration enhancer. In someembodiments, a first phase comprises at least one oligomeric compoundand at least one penetration enhancer, while a second phase comprises atleast one penetration enhancer. In some embodiments, a first phasecomprises at least one oligomeric compound and at least one penetrationenhancer, while a second phase comprises at least one penetrationenhancer and substantially no oligomeric compound. In some embodiments,at least one phase is compounded with at least one degradationretardant, such as a coating or a matrix, which delays release of thecontents of that phase. In some embodiments, a first phase comprises atleast one oligomeric compound, at least one penetration enhancer, whilea second phase comprises at least one penetration enhancer and arelease-retardant. In particular embodiments, an oral oligomericcompound comprises a first phase comprising particles containing anoligomeric compound and a penetration enhancer, and a second phasecomprising particles coated with a release-retarding agent andcontaining penetration enhancer.

A variety of bile salts also function as penetration enhancers tofacilitate the uptake and bioavailability of drugs. The physiologicalroles of bile include the facilitation of dispersion and absorption oflipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etat., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus, the term “bile salt” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579).

In some embodiments, penetration enhancers useful in some embodiments ofpresent invention are mixtures of penetration enhancing compounds. Onesuch penetration enhancer is a mixture of UDCA (and/or CDCA) with capricand/or lauric acids or salts thereof e.g. sodium. Such mixtures areuseful for enhancing the delivery of biologically active substancesacross mucosal membranes, in particular intestinal mucosa. Otherpenetration enhancer mixtures comprise about 5-95% of bile acid orsalt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid.Particular penetration enhancers are mixtures of the sodium salts ofUDCA, capric acid and lauric acid in a ratio of about 1:2:2respectively. Another such penetration enhancer is a mixture of capricand lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (molebasis). In particular embodiments capric acid and lauric acid arepresent in molar ratios of e.g. about 0.1:1 to about 1:0.1, inparticular about 0.5:1 to about 1:0.5.

Other excipients include chelating agents, i.e. compounds that removemetallic ions from solution by forming complexes therewith, with theresult that absorption of oligomeric compounds through the alimentaryand other mucosa is enhanced. With regard to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315).Chelating agents of the invention include, but are not limited to,disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. ControlRet., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers maybe defined as compounds that demonstrate insignificant activity aschelating agents or as surfactants but that nonetheless enhanceabsorption of oligomeric compounds through the alimentary and othermucosal membranes (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1). This class of penetration enhancersincludes, but is not limited to, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligomeric compounds at the cellular levelmay also be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), can be used.

Some oral oligomeric compound compositions also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which may beinert (i.e., does not possess biological activityper se) or may benecessary for transport, recognition or pathway activation or mediation,or is recognized as a nucleic acid by in vivo processes that reduce thebioavailability of an oligomeric compound having biological activity by,for example, degrading the biologically active oligomeric compound orpromoting its removal from circulation. The coadministration of aoligomeric compound and a carrier compound, typically with an excess ofthe latter substance, can result in a substantial reduction of theamount of oligomeric compound recovered in the liver, kidney or otherextracirculatory reservoirs, presumably due to competition between thecarrier compound and the oligomeric compound for a common receptor. Forexample, the recovery of a partially phosphorothioate oligomericcompound in hepatic tissue can be reduced when it is coadministered withpolyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & NuclAcid Drug Dev., 1996, 6, 177).

A “pharmaceutical carrier” or “excipient” may be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more oligomeric compounds to ananimal. The excipient may be liquid or solid and is selected, with theplanned manner of administration in mind, so as to provide for thedesired bulk, consistency, etc., when combined with an oligomericcompound and the other components of a given pharmaceutical composition.Typical pharmaceutical carriers include, but are not limited to, bindingagents (e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and othersugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate,ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB);and wetting agents (e.g., sodium lauryl sulphate, etc.).

Oral oligomeric compound compositions may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipuritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecomposition of present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The oligomeric compounds and compositions of the present invention maybe formulated into any of many possible dosage forms such as, but notlimited to, tablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. The compositions of the present invention mayalso be formulated as suspensions in aqueous, non-aqueous or mixedmedia. Aqueous suspensions may further contain substances which increasethe viscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are described inU.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged nucleic acid molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap nucleic acids rather than complex with it. Both cationic andnoncationic liposomes have been used to deliver nucleic acids andoligomeric compounds to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Formulations for topical administration include those in which theoligomeric compounds of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Lipids and liposomesinclude neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligomeric compounds andcompositions of the invention may be encapsulated within liposomes ormay form complexes thereto, in particular to cationic liposomes.Alternatively, they may be complexed to lipids, in particular tocationic lipids. Topical formulations are described in detail in U.S.patent application Ser. No. 09/315,298 filed on May 20, 1999, which isincorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Oral formulations are thosein which oligomeric compounds of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. A particularly suitable combination is the sodium salt oflauric acid, capric acid and UDCA. Penetration enhancers also includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Compounds and compositions of the invention may be delivered orally, ingranular form including sprayed dried particles, or complexed to formmicro or nanoparticles. Certain oral formulations for oligonucleotidesand their preparation are described in detail in U.S. application Ser.No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,1999) and U.S. Application Publication 20030027780, each of which isincorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more of the compounds and compositions of theinvention and one or more other chemotherapeutic agents that function bya non-antisense mechanism. Examples of such chemotherapeutic agentsinclude but are not limited to cancer chemotherapeutic drugs such asdaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the oligomeric compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of oligomeric compounds and compositions of the inventionand other drugs are also within the scope of this invention. Two or morecombined compounds such as two oligomeric compounds or one oligomericcompound combined with further compounds may be used together orsequentially.

In another embodiment, compositions of the invention may contain one ormore of the compounds and compositions of the invention targeted to afirst nucleic acid target and one or more additional oligomericcompounds targeted to a second nucleic acid target. Alternatively,compositions of the invention may contain two or more oligomericcompounds and compositions targeted to different regions, segments orsites of the same target. Two or more combined compounds may be usedtogether or sequentially.

The formulation of therapeutic compounds and compositions of theinvention and their subsequent administration (dosing) is believed to bewithin the skill of those in the art. Dosing is dependent on severityand responsiveness of the disease state to be treated, with the courseof treatment lasting from several days to several months, or until acure is effected or a diminution of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Persons of ordinary skill caneasily determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages may vary depending on the relative potency ofindividual oligomeric compounds, and can generally be estimated based onEC₅₀s found to be effective in in vitro and in vivo animal models. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, from 0.1μg to 10 g per kg of body weight, from 1.0 μg to 1 g per kg of bodyweight, from 10.0 μg to 100 mg per kg of body weight, from 100 μg to 10mg per kg of body weight, or from 1 mg to 5 mg per kg of body weight,and may be given once or more daily, weekly, monthly or yearly, or evenonce every 2 to 20 years. Persons of ordinary skill in the art caneasily determine repetition rates for dosing based on measured residencetimes and concentrations of the drug in bodily fluids or tissues.Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the oligomeric compound is administered in maintenancedoses, ranging from 0.01 μg to 100 g per kg of body weight, from 0.1 μgto 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight,from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kgof body weight, or from 100 μg to 1 mg per kg of body weight, once ormore daily, to once every 20 years. The effects of treatments withtherapeutic compositions can be assessed following collection of tissuesor fluids from a patient or subject receiving said treatments. It isknown in the art that a biopsy sample can be procured from certaintissues without resulting in detrimental effects to a patient orsubject. In certain embodiments, a tissue and its constituent cellscomprise, but are not limited to, blood (e.g., hematopoietic cells, suchas human hematopoietic progenitor cells, human hematopoietic stem cells,CD34⁺ cells CD4⁺ cells), lymphocytes and other blood lineage cells, bonemarrow, breast, cervix, colon, esophagus, lymph node, muscle, peripheralblood, oral mucosa and skin. In other embodiments, a fluid and itsconstituent cells comprise, but are not limited to, blood, urine, semen,synovial fluid, lymphatic fluid and cerebro-spinal fluid. Tissues orfluids procured from patients can be evaluated for expression levels ofa target small non-coding RNA, mRNA or protein. Additionally, the mRNAor protein expression levels of other genes known or suspected to beassociated with the specific disease state, condition or phenotype canbe assessed. mRNA levels can be measured or evaluated by real-time PCR,Northern blot, in situ hybridization or DNA array analysis.

Protein levels of a downstream target modulated or regulated by a smallnon-coding RNA can be evaluated or quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA),quantitative protein assays, protein activity assays (for example,caspase activity assays), immunohistochemistry, immunocytochemistry orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Western Blot Analysis of Protein Levels:

When small non-coding RNAs have effects on expression of downstreamgenes or proteins encoded by genes, it is advantageous to measure theprotein levels of those gene products. To do this, western blot analysismay be employed.

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligomeric compoundtreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel.Gradient gels (4-20%) may also be used for the separation of proteins,as is known in the art. Gels are typically run for 1.5 hours at 150 V,and transferred to a membrane, such as PVDF, for western blotting.Appropriate primary antibody directed to a target is used, with aradiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Furthermore, the effects of treatment can be assessed by measuringbiomarkers associated with the disease or condition in theaforementioned tissues and fluids, collected from a patient or subjectreceiving treatment, by routine clinical methods known in the art. Thesebiomarkers include but are not limited to: glucose, cholesterol,lipoproteins, triglycerides, free fatty acids and other markers ofglucose and lipid metabolism; liver transaminases, bilirubin, albumin,blood urea nitrogen, creatine and other markers of kidney and liverfunction; interleukins, tumor necrosis factors, intracellular adhesionmolecules, C-reactive protein and other markers of inflammation;testosterone, estrogen and other hormones; tumor markers; vitamins,minerals and electrolytes.

In vitro and in vivo Assays:

Phenotypic Assays

Once modulators are designed or identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive orsuggestive of efficacy in the treatment, amelioration or improvement ofphysiologic conditions associated with a particular disease state orcondition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF7 cells selected for breast cancerstudies; adipocytes for obesity studies) are treated with an oligomericcompound identified from the in vitro studies as well as controlcompounds at optimal concentrations which are determined by the methodsdescribed above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the oligomeric compound.Hallmark genes, or those genes suspected to be associated with aspecific disease state, condition, or phenotype, are measured in bothtreated and untreated cells.

Cell Proliferation and Survival Assays:

In some embodiments, cell proliferation and survival assays are used.Cell cycle regulation is the basis for many cancer therapeutic agents.Unregulated cell proliferation is a characteristic of cancer cells, thusmost current chemotherapy agents target dividing cells, for example, byblocking the synthesis of new DNA required for cell division. However,cells in healthy tissues are often also affected by agents that modulatecell proliferation.

In some cases, a cell cycle inhibitor will cause apoptosis in cancercells, but allow normal cells to undergo growth arrest and thereforeremain unaffected (Blagosklonny, Bioessays, 1999, 21, 704-709; Chen etal., Cancer Res., 1997, 57, 2013-2019; Evan and Littlewood, Science,1998, 281, 1317-1322; Lees and Weinberg, Proc. Natl. Acad. Sci. USA,1999, 96, 4221-4223). An example of sensitization to anti-cancer agentsis observed in cells that have reduced or absent expression of the tumorsuppressor genes p53 (Bunz et al., Science, 1998, 282, 1497-1501; Bunzet al., J. Clin. Invest., 1999, 104, 263-269; Stewart et al., CancerRes., 1999, 59, 3831-3837; Wahl et al., Nat. Med., 1996, 2, 72-79).However, cancer cells often escape apoptosis (Lowe and Lin,Carcinogenesis, 2000, 21, 485-495; Reed, Cancer J. Sci. Am., 1998, 4Suppl 1, S8-14). Further disruption of cell cycle checkpoints in cancercells can increase sensitivity to chemotherapy while allowing normalcells to take refuge in G1 and remain unaffected.

Cell Cycle Assay:

A cell cycle assay is employed to identify genes whose modulationaffects cell cycle progression. In addition to normal cells, cellslacking functional p53 are utilized to identify genes whose modulationwill sensitize p53-deficient cells to anti-cancer agents. Oligomericcompounds of the invention are tested for their effects on the cellcycle in normal human mammary epithelial cells (HMECs) as well as thebreast carcinoma cell lines MCF7 and T47D. The latter two cell linesexpress similar genes but MCF7 cells express the tumor suppressor p53,while T47D cells are deficient in p53. A 20-nucleotide oligomericcompound with a randomized sequence may be used as a negative control,as it does not target modulators of cell cycle progression. Anoligomeric compound targeting kinesin-like 1 is known to inhibit cellcycle progression and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of200 nM of oligomeric compound and 6 μg/mL LIPOFECTIN™. Compounds of theinvention and the positive control are tested in triplicate. Thenegative control is tested in up to six replicate wells. Untreatedcontrol cells receive LIPOFECTIN™ only. Approximately 24, 48 or 72 hoursfollowing transfection, routine procedures are used to prepare cells forflow cytometry analysis and cells are stained with propidium iodide togenerate a cell cycle profile using a flow cytometer. The cell cycleprofile is analyzed with the ModFit program (Verity Software House,Inc., Topsham Me.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces anincrease in cells with a hypodiploid DNA content, which are categorizedas “subG1.” An increase in cells in G1 phase is indicative of a cellcycle arrest prior to entry into S phase; an increase in cells in Sphase is indicative of cell cycle arrest during DNA synthesis; and anincrease in cells in the G2/M phase is indicative of cell cycle arrestjust prior to or during mitosis. Cell cycle profiles of cells treatedwith oligomeric compounds can be normalized to those of untreatedcontrol cells, and values above or below 100% are considered to indicatean increase or decrease, respectively, in the proportion of cells in aparticular phase of the cell cycle.

Oligomeric compounds that prevent cell cycle progression are candidatetherapeutic agents for the treatment of hyperproliferative disorders,such as cancer or inflammation.

Caspase Assay:

Programmed cell death, or apoptosis, is an important aspect of variousbiological processes, including normal cell turnover, immune systemdevelopment and embryonic development. Apoptosis involves the activationof caspases, a family of intracellular proteases through which a cascadeof events leads to the cleavage of a select set of proteins. The caspasefamily can be divided into two groups: the initiator caspases, such ascaspase-8 and -9, and the executioner caspases, such as caspase-3, -6and -7, which are activated by the initiator caspases. The caspasefamily contains at least 14 members, with differing substratepreferences (Thomberry and Lazebnik, Science, 1998, 281, 1312-1316). Acaspase assay is utilized to identify genes whose modulation causesapoptosis. The chemotherapeutic drugs taxol, cisplatin, etoposide,gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have beenshown to induce apoptosis in a caspase-dependent manner.

In a further embodiment, a caspase assay is employed to identify genesor targets whose modulation affects apoptosis. In addition to normalcells, cells lacking functional p53 are utilized to identify genes ortargets whose modulation will sensitize p53-deficient cells to agentsthat induce apoptosis. Oligomeric compounds of the invention are assayedfor their affects on apoptosis in normal HMECs as well as the breastcarcinoma cell lines MCF7 and T47D. HMECs and MCF7 cells express p53,whereas T47D cells do not express this tumor suppressor gene. Cells arecultured in 96-well plates with black sides and flat, transparentbottoms (Corning Incorporated, Corning, N.Y.). DMEM medium, with andwithout phenol red, is obtained from Invitrogen Life Technologies(Carlsbad, Calif.). MEGM medium, with and without phenol red, isobtained from Cambrex Bioscience (Walkersville, Md.). A 20-nucleotideoligomeric compound with a randomized sequence may be used as a negativecontrol, as it does not target modulators of caspase activity. Anoligomeric compound targeted to human Jagged2 or human Notch1, both ofwhich are known to induce caspase activity, may be used as a positivecontrol for caspase activation.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of200 nM of oligomeric compound and 6 μg/mL LIPOFECTIN™. Compounds of theinvention and the positive controls are tested in triplicate, and thenegative control is tested in up to six replicate wells. Untreatedcontrol cells receive LIPOFECTIN™ only.

Caspase-3 activity is evaluated with a fluorometric HTS Caspase-3 assay(Catalog #HTS02; EMD Biosciences, San Diego, Calif.) that detectscleavage after aspartate residues in the peptide sequence DEVD. The DEVDsubstrate is labeled with a fluorescent molecule, which exhibits a blueto green shift in fluorescence upon cleavage by caspase-3. Activecaspase-3 in the oligomeric compound-treated cells is measured by thisassay according to the manufacturer's instructions. Approximately 48hours following treatment, 50 μL of assay buffer containing 10 μMdithiothreitol is added to each well, followed by addition 20 μL of thecaspase-3 fluorescent substrate conjugate. Fluorescence in wells isimmediately detected (excitation/emission 400/505 nm) using afluorescent plate reader (SpectraMAX GeminiXS, Molecular Devices,Sunnyvale, Calif.). The plate is covered and incubated at 37° C. for anadditional three hours, after which the fluorescence is again measured(excitation/emission 400/505 nm). The value at time zero is subtractedfrom the measurement obtained at 3 hours. The measurement obtained fromthe untreated control cells is designated as 100% activity. Caspase-3activity in cells treated with oligomeric compounds is normalized tothat in untreated control cells. Values for caspase activity above orbelow 100% are considered to indicate that the compound has the abilityto stimulate or inhibit caspase activity, respectively.

Oligomeric compounds that cause a significant induction in apoptosis arecandidate therapeutic agents with applications in the treatment ofconditions in which the induction of apoptosis is desirable, forexample, in hyperproliferative disorders. Oligomeric compounds thatinhibit apoptosis are candidate therapeutic agents with applications inthe treatment of conditions where the reduction of apoptosis is useful,for example, in neurodegenerative disorders.

Angiogenesis Assays:

In some embodiments, angiogenesis assays are used. Angiogenesis is thegrowth of new blood vessels (veins and arteries) by endothelial cells.This process is important in the development of a number of humandiseases, and is believed to be particularly important in regulating thegrowth of solid tumors. Without new vessel formation it is believed thattumors will not grow beyond a few millimeters in size. In addition totheir use as anti-cancer agents, inhibitors of angiogenesis havepotential for the treatment of diabetic retinopathy, cardiovasculardisease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature,2000, 407, 249-257; Freedman and Isner, J. Mol. Cell Cardiol, 2001, 33,379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al.,Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine,2000, 67, 366-383; Yoshida et al., Histol Histopathol., 1999, 14,1287-1294).

Expression of Angiogenic Genes as a Measure of Angiogenesis:

During the process of angiogenesis, endothelial cells perform severaldistinct functions, including the degradation of the extracellularmatrix (ECM), migration, proliferation and the formation of tube-likestructures (Liekens et al., Biochem. Pharmacol, 2001, 61, 253-270).Endothelial cells must regulate the expression of many genes in order toperform the functions necessary for angiogenesis. This gene regulationhas been the subject of intense scrutiny, and many genes have beenidentified as being important for the angiogenic phenotype. Genes highlyexpressed in angiogenic endothelial cells include integrin β3,endoglin/CD105, TEM5 and MMP-14/MT-MMP1.

Integrin β3 is part of a family of heterodimeric transmembrane receptorsthat consist of alpha and beta subunits (Brooks et al., J. Clin.Invest., 1995, 96, 1815-1822). Each subunit recognizes a unique set ofECM ligands, thereby allowing cells to transmit angiogenic signals fromthe extracellular matrix. Integrin β3 is prominently expressed onproliferating vascular endothelial cells, and it plays roles in allowingnew blood vessels to form at tumor sites as well as allowing theepithelial cells of breast tumors to spread (Brooks et al., J. Clin.Invest., 1995, 96, 1815-1822; Drake et al., J. Cell Sci., 1995, 108 (Pt7), 2655-2661). Blockage of integrin β3 with monoclonal antibodies orlow molecular weight antagonists inhibits blood vessel formation in avariety of in-vivo models, including tumor angiogenesis andneovascularization during oxygen-induced retinopathy (Brooks et al.,Science, 1994, 264, 569-571; Brooks et al., J. Clin. Invest., 1995, 96,1815-1822; Hammes et al., Nat. Med., 1996, 2, 529-533).

Endoglin is a transforming growth factor receptor-associated proteinhighly expressed on endothelial cells, and present on some leukemiacells and minor subsets of bone marrow cells (Burrows et al., Clin.Cancer Res., 1995, 1, 1623-1634; Haruta and Seon, Proc. Natl. Acad. Sci.US A, 1986, 83, 7898-7902). Its expression is upregulated in endothelialcells of angiogenic tissues and is therefore used as a prognosticindicator in various tumors (Burrows et al., Clin. Cancer Res., 1995, 1,1623-1634). Endoglin functions as an ancillary receptor influencingbinding of the transforming growth factor beta (TGF-beta) family ofligands to signaling receptors, thus mediating cell survival (Massagueand Chen, Genes Dev., 2000, 14, 627-644).

Tumor endothelial marker 5 (TEM5) is a putative 7-pass transmembraneprotein (GPCR) (Carson-Walter et al., Cancer Res., 2001, 61, 6649-6655).The mRNA transcript, designated KIAA1531, encodes one of many tumorendothelium markers (TEMs) that display elevated expression (greaterthan 10-fold) during tumor angiogenesis (St Croix et al., Science, 2000,289, 1197-1202). TEM5 is coordinately expressed with other TEMs on tumorendothelium in humans and mice.

Matrix metalloproteinase 14 (MMP-14), a membrane-type MMP covalentlylinked to the cell membrane, is involved in matrix detachment andmigration. MMP-14 is thought to promote tumor angiogenesis; antibodiesdirected against the catalytic domain of MMP-14 block endothelial-cellmigration, invasion and capillary tube formation in vitro (Galvez etal., J. Biol. Chem., 2001, 276, 37491-37500). MMP-14 can degrade thefibrin matrix that surrounds newly formed vessels potentially allowingthe endothelial cells to invade further into the tumor tissue (Hotary etal., J. Exp. Med., 2002, 195, 295-308). MMP-14 null mice have impairedangiogenesis during development, further demonstrating the role ofMMP-14 in angiogenesis (Vu and Werb, Genes Dev., 2000, 14, 2123-2133;Zhou et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 4052-4057).

In some embodiments, HUVECs are used to measure the effects ofoligomeric compounds of the invention on the activity of endothelialcells stimulated with human vascular endothelial growth factor (VEGF). A20-nucleotide oligomeric compound with a randomized sequence may be usedas a negative control, as it does not target modulators of HUVECactivity.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Compounds ofthe invention are tested in triplicate, and the negative control istested in up to six replicate wells. Untreated control cells receiveLIPOFECTIN™only.

Approximately twenty hours after transfection, cells are induced toexpress angiogenic genes with recombinant VEGF. Total RNA is harvestedapproximately 52 hours following transfection, and the amount of totalRNA from each sample is determined using a Ribogreen Assay (InvitrogenLife Technologies, Carlsbad, Calif.). Real-time RT-PCR is performed onthe total RNA using primer/probe sets for four angiogenic hallmark genesdescribed herein: integrin β3, endoglin, TEM5 and MMP14. Expressionlevels for each gene are normalized to total RNA. Gene expression incells treated with oligomeric compounds is normalized to that inuntreated control cells. A value above or below 100% is considered toindicated an increase or decrease in gene expression, respectively.

Oligomeric compounds resulting in a decrease in the expression ofangiogenic hallmark genes are candidate therapeutic agents for theinhibition of angiogenesis where such activity is desired, for example,in the treatment of cancer, diabetic retinopathy, cardiovasculardisease, rheumatoid arthritis and psoriasis. Oligomeric compounds thatincrease the expression of angiogenic hallmark genes are candidatetherapeutic agents with applications where the stimulation ofangiogenesis is desired, for example, in wound healing.

Endothelial Tube Formation Assay as a Measure of Angiogenesis:

Angiogenesis is stimulated by numerous factors that promote interactionof endothelial cells with each other and with extracellular matrixmolecules, resulting in the formation of capillary tubes. Thismorphogenic process is necessary for the delivery of oxygen to nearbytissues and plays an essential role in embryonic development, woundhealing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407,249-257). Moreover, this process can be reproduced in a tissue cultureassay that evaluated the formation of tube-like structures byendothelial cells. There are several different variations of the assaythat use different matrices, such as collagen I (Kanayasu et al.,Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem.,1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998,238, 324-334), as growth substrates for the cells. In this assay, HUVECsare plated on a matrix derived from the Engelbreth-Holm-Swarm mousetumor, which is very similar to Matrigel (Kleinman et al., Biochemistry,1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34,85-91). Untreated HUVECs form tube-like structures when grown on thissubstrate. Loss of tube formation in vitro has been correlated with theinhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000,407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), whichsupports the use of in vitro tube formation as an endpoint forangiogenesis.

In some embodiments, HUVECs are used to measure the effects ofoligomeric compounds of the invention on endothelial tube formationactivity. The tube formation assay is performed using an in vitroAngiogenesis Assay Kit (Chemicon International, Temecula, Calif.). A20-nucleotide oligomeric compound with a randomized sequence may be usedas a negative control, as it does not target modulators of endothelialtube formation.

Oligomeric compounds are mixed with LIPOFECTIN™ in OPTI-MEM™ to achievea final concentration of 75 nM of oligomeric compound and 2.25 μg/mLLIPOFECTIN™. Untreated control cells receive LIPOFECTIN™ only. Compoundsof the invention are tested in triplicate, and the negative control istested in up to six replicates.

Approximately fifty hours after transfection, cells are transferred to96-well plates coated with ECMatrix™ (Chemicon International). Underthese conditions, untreated HUVECs form tube-like structures. After anovernight incubation at 37° C., treated and untreated cells areinspected by light microscopy. Tube formation in cells treated witholigomeric compounds is compared to that in untreated control cells.Individual wells are assigned discrete scores from 1 to 5 depending onthe extent of tube formation. A score of 1 refers to a well with no tubeformation while a score of 5 is given to wells where all cells areforming an extensive tubular network.

Oligomeric compounds resulting in a decrease in tube formation arecandidate therapeutic agents for the inhibition of angiogenesis wheresuch activity is desired, for example, in the treatment of cancer,diabetic retinopathy, cardiovascular disease, rheumatoid arthritis andpsoriasis. Oligomeric compounds that promote endothelial tube formationare candidate therapeutic agents with applications where the stimulationof angiogenesis is desired, for example, in wound healing.

Matrix Metalloproteinase Activity:

During angiogenesis, endothelial cells must degrade the extracellularmatrix (ECM) and thus secrete matrix metalloproteinases (MMPs) in orderto accomplish this degradation. MMPs are a family of zinc-dependentendopeptidases that fall into eight distinct classes: five are secretedand three are membrane-type MMPs (MT-MMPs) (Egeblad and Werb, J. CellScience, 2002, 2, 161-174). MMPs exert their effects by cleaving adiverse group of substrates, which include not only structuralcomponents of the extracellular matrix, but also growth-factor-bindingproteins, growth-factor pre-cursors, receptor tyrosine-kinases,cell-adhesion molecules and other proteinases (Xu et al., J. Cell Biol.,2002, 154, 1069-1080).

In some embodiments, oligomeric compounds of the invention are evaluatedfor their effects on MMP activity in the medium above cultured HUVECs.MMP activity is measured using the EnzChek Gelatinase/Collagenase AssayKit (Molecular Probes, Eugene, Oreg.). In this assay, HUVECs are platedat approximately 4000 cells per well in 96-well plates and transfectedone day later. A 20-nucleotide oligomeric compound with a randomizedsequence may be used as a negative control, as it does not targetmodulators of MMP activity. An oligomeric compound targeted to integrinβ3 is known to inhibit MMP activity and may be used as a positivecontrol.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Compounds ofthe invention and the positive control are tested in triplicate, and thenegative control is tested in up to six replicates. Untreated controlcells receive LIPOFECTIN™ only.

Approximately 50 hours after transfection, a p-aminophenylmercuricacetate (APMA, Sigma-Aldrich, St. Louis, Mo.) solution is added to eachwell of a Corning-Costar 96-well clear bottom plate (VWR International,Brisbane, Calif.). The APMA solution is used to promote cleavage ofinactive MMP precursor proteins. Medium above the HUVECs is thentransferred to the wells in the 96-well plate. After approximately 30minutes, the quenched, fluorogenic MMP cleavage substrate is added, andbaseline fluorescence is read immediately at 485 nm excitation/530 nmemission. Following an overnight incubation at 37° C. in the dark,plates are read again to determine the amount of fluorescence, whichcorresponds to MMP activity. Total protein from HUVEC lysates is used tonormalize the readings, and MMP activity from cells treated witholigomeric compounds is normalized to that of untreated control cells.MMP activities above or below 100% are considered to indicate astimulation or inhibition, respectively, of MMP activity.

Oligomeric compounds resulting in a decrease in MMP activity arecandidate therapeutic agents for the inhibition of angiogenesis wheresuch activity is desired, for example, in the treatment of cancer,diabetic retinopathy, cardiovascular disease, rheumatoid arthritis andpsoriasis. Oligomeric compounds that increase the expression ofangiogenic hallmark genes are candidate therapeutic agents withapplications in conditions requiring angiogenesis, for example, in woundhealing.

Adipocyte Assays:

In some embodiments, adipocytes assays are used. Insulin is an essentialsignaling molecule throughout the body, but its major target organs arethe liver, skeletal muscle and adipose tissue. Insulin is the primarymodulator of glucose homeostasis and helps maintain a balance ofperipheral glucose utilization and hepatic glucose production. Thereduced ability of normal circulating concentrations of insulin tomaintain glucose homeostasis manifests in insulin resistance which isoften associated with diabetes, central obesity, hypertension,polycystic ovarian syndrome, dyslipidemia and atherosclerosis (Saltiel,Cell, 2001, 104, 517-529; Saltiel and Kahn, Nature, 2001, 414, 799-806).

Response of Undifferentiated Adipocytes to Insulin:

Insulin promotes the differentiation of preadipocytes into adipocytes.The condition of obesity, which results in increases in fat cell number,occurs even in insulin-resistant states in which glucose transport isimpaired due to the antilipolytic effect of insulin. Inhibition oftriglyceride breakdown requires much lower insulin concentrations thanstimulation of glucose transport, resulting in maintenance or expansionof adipose stores (Kitamura et al., Mol Cell Biol., 1999, 19, 6286-6296;Kitamura et al., Mol Cell Biol., 1998, 18, 3708-3717).

One of the hallmarks of cellular differentiation is the upregulation ofgene expression. During adipocyte differentiation, the gene expressionpatterns in adipocytes change considerably. Some genes known to beupregulated during adipocyte differentiation include hormone-sensitivelipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter4 (Glut4), and peroxisome proliferator-activated receptor gamma(PPAR-γ). Insulin signaling is improved by compounds that bind andinactivate PPAR-γ, a key regulator of adipocyte differentiation(Olefsky, J. Clin. Invest., 2000, 106, 467-472). Insulin induces thetranslocation of GLUT4 to the adipocyte cell surface, where ittransports glucose into the cell, an activity necessary for triglyceridesynthesis. In all forms of obesity and diabetes, a major factorcontributing to the impaired insulin-stimulated glucose transport inadipocytes is the downregulation of GLUT4. Insulin also induces hormonesensitive lipase (HSL), which is the predominant lipase in adipocytesthat functions to promote fatty acid synthesis and lipogenesis(Fredrikson et al., J. Biol. Chem., 1981, 256, 6311-6320). Adipocytefatty acid binding protein (aP2) belongs to a multi-gene family of fattyacid and retinoid transport proteins. aP2 is postulated to serve as alipid shuttle, solubilizing hydrophobic fatty acids and delivering themto the appropriate metabolic system for utilization (Fu et al., J. LipidRes., 2000, 41, 2017-2023; Pelton et al., Biochem. Biophys. Res.Commun., 1999, 261, 456-458). Together, these genes play important rolesin the uptake of glucose and the metabolism and utilization of fats.

Leptin secretion and an increase in triglyceride content are alsowell-established markers of adipocyte differentiation. In addition toits role in adipocytes differentiation, leptin also regulates glucosehomeostasis through mechanisms (autocrine, paracrine, endocrine andneural) independent of the adipocyte's role in energy storage andrelease. As adipocytes differentiate, insulin increases triglycerideaccumulation by both promoting triglyceride synthesis and inhibitingtriglyceride breakdown (Spiegelman and Flier, Cell, 2001, 104, 531-543).As triglyceride accumulation correlates tightly with cell size and cellnumber, it is an excellent indicator of differentiated adipocytes.

Oligomeric compounds of the invention are tested for their effects onpreadipocyte differentiation. A 20-nucleotide oligomeric compound with arandomized sequence may be used as a negative control, as it does nottarget modulators of adipocyte differentiation. Tumor necrosis factoralpha (TNF-α) is known to inhibit adipocyte differentiation and may beused as a positive control for the inhibition of adipocytedifferentiation as evaluated by leptin secretion. For the otheradipocyte differentiation markers assayed, an oligomeric compoundtargeted to PPAR-γ, also known to inhibit adipocyte differentiation, maybe used as a positive control.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of250 nM of oligomeric compound and 7.5 μg/mL LIPOFECTIN™. Untreatedcontrol cells receive LIPOFECTIN™ only. Compounds of the invention andthe positive control are tested in triplicate, and the negative controlis tested in up to six replicate wells.

After the cells have reach confluence (approximately three days), theyare exposed for an additional three days to differentiation medium(Zen-Bio, Inc., Research Triangle Park, N.C.) containing a PPAR-γagonist, IBMX, dexamethasone, and insulin. Cells are then fed adipocytemedium (Zen-Bio, Inc.), which is replaced at 2 or 3 day intervals.

Leptin secretion into the medium in which adipocytes are cultured ismeasured by protein ELISA. On day nine post-transfection, 96-well platesare coated with a monoclonal antibody to human leptin (R&D Systems,Minneapolis, Minn.) and left at 4° C. overnight. The plates are blockedwith bovine serum albumin (BSA), and a dilution of the treated adipoctyemedium is incubated in the plate at room temperature for approximately 2hours. After washing to remove unbound components, a second monoclonalantibody to human leptin (conjugated with biotin) is added. The plate isthen incubated with strepavidin-conjugated horse radish peroxidase (HRP)and enzyme levels are determined by incubation with3,3′,5,5′-tetramethlybenzidine, which turns blue when cleaved by HRP.The OD₄₅₀ is read for each well, where the dye absorbance isproportional to the leptin concentration in the cell lysate. Leptinsecretion from cells treated with oligomeric compounds is normalized tothat from untreated control cells. With respect to leptin secretion,values above or below 100% are considered to indicate that the compoundhas the ability to stimulate or inhibit leptin secretion, respectively.

The triglyceride accumulation assay measures the synthesis oftriglyceride by adipocytes. Triglyceride accumulation is measured usingthe Infinity™ Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.).On day nine post-transfection, cells are washed and lysed at roomtemperature, and the triglyceride assay reagent is added. Triglycerideaccumulation is measured based on the amount of glycerol liberated fromtriglycerides by the enzyme lipoprotein lipase. Liberated glycerol isphosphorylated by glycerol kinase, and hydrogen peroxide is generatedduring the oxidation of glycerol-1-phosphate to dihydroxyacetonephosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP)uses H₂O₂ to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzenesulfonate to produce a red-colored dye. Dye absorbance, which isproportional to the concentration of glycerol, is measured at 515 nmusing an UV spectrophotometer. Glycerol concentration is calculated froma standard curve for each assay, and data are normalized to totalcellular protein as determined by a Bradford assay (Bio-RadLaboratories, Hercules, Calif.). Triglyceride accumulation in cellstreated with oligomeric compounds is normalized to that in untreatedcontrol cells. Values for triglyceride accumulation above or below 100%are considered to indicate that the compound has the ability tostimulate or inhibit triglyceride accumulation, respectively.

Expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, isalso measured in adipocytes transfected with oligomeric compounds of theinvention. Cells are lysed on day nine post-transfection and total RNAis harvested. The amount of total RNA in each sample is determined usinga Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.).Real-time PCR is performed on the total RNA using primer/probe sets forthe adipocyte differentiation hallmark genes Glut4, HSL, aP2, andPPAR-Y. Gene expression in cells treated with oligomeric compounds isnormalized to that in untreated control cells. With respect to the fouradipocyte differentiation hallmark genes, values above or below 100% areconsidered to indicate that the compound has the ability to stimulate orinhibit adipocyte differentiation, respectively.

Oligomeric compounds that reduce the expression levels of markers ofadipocyte differentiation are candidate therapeutic agents withapplications in the treatment, attenuation or prevention of obesity,hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension,or other metabolic diseases as well as having potential applications inthe maintenance of the pluripotent phenotype of stem or precursor cells.Oligomeric compounds of the invention resulting in a significantincrease in leptin secretion are potentially useful for the treatment ofobesity.

Response of Liver-Derived Cells to Insulin:

Insulin mediates its effects by suppressing the RNA expression levels ofenzymes important for gluconeogenesis and glycogenolysis, and also bycontrolling the activities of some metabolic enzymes throughpost-translational mechanisms (Hall and Granner, J. Basic Clin. PhysiolPharmacol, 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827;Saltiel and Kahn, Nature, 2001, 414, 799-806). In liver cells, genesinvolved in regulating glucose metabolism can be identified bymonitoring changes in the expression of selective insulin-responsivegenes in a cell culture model. However, primary human hepatocytes aredifficult to obtain and work with in culture. Therefore, the insulinsignaling assay described herein is performed in the hepatocellularcarcinoma cell line HepG2, the most widely used cell culture model forhepatocytes. The insulin responsive genes evaluate in this assay arephosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factorbinding protein 1 (IGFBP-1) and follistatin.

IGFBP-1 is one of a family of six secreted proteins that bindinsulin-like growth factor (IGF) with high affinity and thereby modulateIGFs action in vivo (Baxter, Am. J. Physiol Endocrinol Metab., 2000,278, E967-976; Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216,319-357). IGFBP-1 is characterized by dynamic variability of levels incirculation due to the regulation of its hepatic secretion (Lee et al.,Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). The multi-hormonalregulation of PEPCK and IGFBP-1 are similar. Glucocorticoids and cyclicAMP (cAMP) stimulate transcription of the IGFBP-1 gene expressionwhereas insulin acts in a dominant manner to suppress both basal andcAMP or glucocorticoid-stimulated IGFBP-1 gene transcription (O'Brienand Granner, Physiol. Rev., 1996, 76, 1109-1161). PEPCK catalyzes therate-limiting step in gluconeogenesis, and thereby contributes tohepatic glucose output (Hall and Granner, J. Basic Clin. PhysiolPharmacol, 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827;Saltiel and Kahn, Nature, 2001, 414, 799-806). In hepatoma cells,studies have shown that the expression of PEPCK is stimulated byglucocorticoids, glucagon (via cAMP), and retinoic acid. Insulin acts ina dominant manner to suppress these stimulations as well as basaltranscription (O'Brien and Granner, Physiol Rev., 1996, 76, 1109-1161).In HepG2 cells, prolonged serum starvation induces the expression ofPEPCK and subsequent insulin stimulation significantly reduces the PEPCKmRNA level.

Follistatin is significantly stimulated by insulin in HepG2 cells.Interestingly, follistatin levels have been shown to be higher in womenwith polycystic ovary syndrome (PCOS) (Norman et al., Hum. Reprod.,2001, 16, 668-672). PCOS is a metabolic as well as a reproductivedisorder, and an important cause of type 2 diabetes mellitus in women.It is often associated with profound insulin resistance andhyperinsulinemia as well as with a defect in insulin secretion (Dunaif,Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil Steril, 2002,77, 209-215). PCOS is the most common cause of female infertility in theU.S. and affects 5%-10% of women of child-bearing age (Dunaif, Endocr.Rev., 1997, 18, 774-800; Nestler et al., Fertil Steril, 2002, 77,209-215).

In some embodiments, HepG2 cells are used to measure the effects ofcompounds of the invention on hepatic gene expression following insulinstimulation. A 20-nucleotide oligomeric compound with a randomizedsequence may be used as a negative control, as it does not targetmodulators of hepatic gene expression. Insulin at a concentration of 100nM may be used as a positive control for the stimulation of hepatic geneexpression. An oligomeric compound targeted to human forkhead is knownto inhibit hepatic gene expression and may be used as a positive controlfor the inhibition of gene expression in the presence of insulin.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of100 nM of oligomeric compound and 3 μg/mL LIPOFECTIN™. Untreated controlcells receive LIPOFECTIN™ only. Compounds of the invention and thepositive controls are tested in triplicate, and the negative control istested in up to six replicate wells.

Approximately 28 hours after transfection, the cells are subjected toserum starvation for a period of 12 to 16 hours, using serum-free growthmedium. Following serum starvation, cells are treated with 1 nM insulin(insulin-treated) or are left untreated (basal conditions) forapproximately four hours. At the same time, untreated control cells inboth plates are treated with 100 nM insulin to determine the maximalinsulin response. Following insulin treatment (forty-eight hours aftertransfection), total RNA is harvested from all samples, and the amountof total RNA from each sample is determined using a Ribogreen assay(Invitrogen Corporation, Carlsbad, Calif.). Real-time PCR is performedon the total RNA samples using primer/probe sets for three insulinresponsive genes: insulin-like growth factor binding protein-1(IGFBP-1), cytosolic PEPCK (PEPCK-C), and follistatin. Gene expressionlevels obtained by real-time PCR are normalized for total RNA content inthe samples. Gene expression in cells treated with oligomeric compoundsis normalized to that from untreated control cells. Values above orbelow 100% are considered to indicate an increase or decrease in geneexpression, respectively.

Oligomeric compounds that interfere with the expression of genesinvolved in glucose metabolism are candidate therapeutic agents for thetreatment of conditions associated with abnormal glucose metabolism, forexample, obesity and diabetes.

Inflammation Assays:

In some embodiments, inflammation assays are used. Inflammation assaysare designed to identify genes that regulate the activation and effectorphases of the adaptive immune response. During the activation phase, Tlymphocytes (also known as T-cells) receiving signals from theappropriate antigens undergo clonal expansion, secrete cytokines, andup-regulate their receptors for soluble growth factors, cytokines andco-stimulatory molecules (Cantrell, Annu. Rev. Immunol, 1996, 14,259-274). These changes drive T-cell differentiation and effectorfunction. Response to cytokines by non-immune effector cells controlsthe production of inflammatory mediators that can do extensive damage tohost tissues. The cells of the adaptive immune systems, their products,as well as their interactions with various enzyme cascades involved ininflammation (e.g., the complement, clotting, fibrinolytic and kinincascades) all represent potential points for intervention ininflammatory disease.

Dendritic cells treated with oligomeric compounds targeting differentgenes are used to identify regulators of dendritic cell-mediated T-cellco-stimulation. The level of interleukin-2 (IL-2) production by T-cells,a critical consequence of T-cell activation (DeSilva et al., J.Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev.Immunol., 2001, 19, 225-252), is used as an endpoint for T-cellactivation. T lymphocytes are important immunoregulatory cells thatmediate pathological inflammatory responses. Optimal activation of Tlymphocytes requires both primary antigen recognition events as well assecondary or co-stimulatory signals from antigen presenting cells (APC).Dendritic cells are the most efficient APCs known and are principallyresponsible for antigen presentation to T-cells, expression of highlevels of co-stimulatory molecules during infection and disease, and theinduction and maintenance of immunological memory (Banchereau andSteinman, Nature, 1998, 392, 245-252). While a number of co-stimulatoryligand-receptor pairs have been shown to influence T-cell activation, aprincipal signal is delivered by engagement of CD28 on T-cells by CD80(B7-1) and CD86 (B7-2) on APCs (Boussiotis et al., Curr. Opin. Immunol,1994, 6, 797-807; Lenschow et al., Annu. Rev. Immunol, 1996, 14,233-258). In contrast, a B7 counter-receptor, CTLA-4, has been shown tonegatively regulate T-cell activation, maintain immunologicalhomeostasis and promote immune tolerance (Walunas and Bluestone, J.Immunol., 1998, 160, 3855-3860). Inhibition of T-cell co-stimulation byAPCs holds promise for novel and more specific strategies of immunesuppression. In addition, blocking co-stimulatory signals may lead tothe development of long-term immunological anergy (unresponsiveness ortolerance) that would offer utility for promoting transplantation ordampening autoimmunity. T-cell anergy is the direct consequence offailure of T-cells to produce the growth factor interleukin-2 (DeSilvaet al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu.Rev. Immunol., 2001, 19, 225-252). Dendritic cell cytokine production asa measure of the activation phase of the immune response:

In some embodiments, the effects of the oligomeric compounds of theinvention are examined on the dendritic cell-mediated costimulation ofT-cells. A 20-nucleotide oligomeric compound with a randomized sequencemay be used as a negative control, as it does not target modulators ofdendritic cell-mediated T-cell costimulation. An oligomeric compoundtargeted to human CD86 is known to inhibit dendritic cell-mediatedT-cell stimulation and may be used as a positive control.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of200 nM of oligomeric compound and 6 μg/mL LIPOFECTIN™. Untreated controlcells receive LIPOFECTIN™ only. Compounds of the invention and thepositive control are tested in triplicate, and the negative control istested in up to six replicates. Following incubation with the oligomericcompounds and LIPOFECTIN™, fresh growth medium with cytokines is addedand DC culture is continued for an additional 48 hours. DCs are thenco-cultured with Jurkat T-cells in RPMI medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% heat-inactivatedfetal bovine serum (Sigma Chemical Company, St. Louis, Mo.). Culturesupernatants are collected 24 hours later and assayed for IL-2 levels(IL-2 DuoSet, R&D Systems, Minneapolis, Minn.). IL-2 levels in cellstreated with oligomeric compounds are normalized to those from untreatedcontrol cells. A value greater than 100% indicates an induction of theinflammatory response, whereas a value less than 100% demonstrates areduction in the inflammatory response.

Oligomeric compounds that inhibit T-cell co-stimulation are candidatetherapeutic compounds with applications in the prevention, treatment orattenuation of conditions associated with hyperstimulation of the immunesystem, including rheumatoid arthritis, irritable bowel disease, asthma,lupus and multiple sclerosis. Oligomeric compounds that induce T-cellco-stimulation are candidate therapeutic agents for the treatment ofimmunodeficient conditions.

Cytokine Signaling as a Measure of the Effector Phase of theInflammatory Response:

The cytokine signaling assay further identifies genes that regulateinflammatory responses of non-immune effector cells (initiallyendothelial cells) to stimulation with cytokines such asinterferon-gamma (IFN-γ). Response to IFN-γ is assessed by measuring theexpression levels of three genes: intercellular adhesion molecule-1(ICAM-1), interferon regulatory factor 1 (IRF1) and small induciblecytokine subfamily B (Cys-X-Cys), member 11 (SCYB11). The cytokinesignaling assay further identifies genes that regulate inflammatoryresponses of non-immune effector cells (initially endothelial cells) tostimulation with IL-1β or TNF-α (Heyninck et al., J Cell Biol, 1999,145, 1471-1482; Zetoune et al., Cytokine, 2001, 15, 282-298). Responseto IL-1β or TNF-α stimulation is monitored by measuring the expressionlevels of four genes: A20, intracellular adhesion molecule 1 (ICAM-1),interleukin-9 (IL-8) and macrophage-inflammatory protein 2 (MIP2α). Asdescribed below, all of these genes regulate numerous parameters of theinflammatory response.

ICAM-1 is an adhesion molecule expressed at low levels on restingendothelial cells that is markedly up-regulated in response toinflammatory mediators like tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β) and interferon-γ (IFN-γ) (Springer, Nature, 1990,346, 425-434). ICAM-1 expression serves to attract circulatingleukocytes into the inflammatory site.

IRF-1 binds to upstream cis-regulatory elements of interferon-induciblegenes and functions as a transcriptional activator. IRF-1 directly bindsto a functional IFN-γ-stimulated response element in the cathepsin Spromoter and mediates IFN-γ dependent transcriptional activation (Stormvan's Gravesande et al., J Immunol., 2002, 168, 4488-4494).

SCYB11 is essential for mediating normal leukocyte recruitment andtrafficking during inflammation. SCYB11 induces a chemotactic responsein IL-2 activated T-cells, monocytes and granulocytes (Mohan et al., JImmunol., 2002, 168, 6420-6428).

A20 is a zinc-finger protein that limits the transcription ofpro-inflammatory genes by blocking TRAF2-stimulated NK-κB signaling.Studies in mice show that TNF-α dramatically increases A20 expression inmice, and that A20 expression is crucial for their survival (Lee et al.,Science, 2000, 289, 2350-2354).

IL-8 is a member of the chemokine gene superfamily, members of whichpromote the pro-inflammatory phenotype of macrophages, vascular smoothmuscle cells and endothelial cells (Koch et al., Science, 1992, 258,1798-1801). IL-8 has been known as one of the major inducible chemokineswith the ability to attract neutrophils to the site of inflammation.More recently, IL-8 has been implicated as a major mediator of acuteneutrophil-mediated inflammation, and is therefore a potentialanti-inflammatory target (Mukaida et al., Cytokine Growth Factor Rev,1998, 9, 9-23).

MIP2α, another chemokine known to play a central role in leukocyteextravasation, has more recently been shown to be involved in acuteinflammation (Lukacs et al., Chem Immunol., 1999, 72, 102-120). MIP2α isexpressed in response to microbial infection, to injection oflipopolysaccharides (LPS), and to stimulation of cells withpro-inflammatory mediators such as IL-1β and TNF-α (Kopydlowski et al.,J Immunol., 1999, 163, 1537-1544). Endothelial cells are one of severalcell types that are sources of MIP2α (Rudner et al., J Immunol., 2000,164, 6576-6582).

In some embodiments, the effects of the oligomeric compounds of theinvention on the cellular response to cytokines may be examined inHUVECs. A 20-nucleotide oligomeric compound with a randomized sequencemay be used as a negative control, as it does not target modulators ofcytokine signaling.

Cells are transfected as described herein. Oligomeric compounds aremixed with LIPOFECTIN™ in OPTI-MEM™ to achieve a final concentration of75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Untreatedcontrol cells receive LIPOFECTIN™ only. Compounds of the invention aretested in triplicate, and the negative control is tested in up to sixreplicate wells.

For IFN-γ stimulation, following transfection, fresh growth medium isadded and DC culture is continued for an additional 44 hours, afterwhich HUVECS are stimulated with 10 ng/ml of IFN-γ for a period of 4hours. For stimulation with IL-1β or TNF-α, fresh growth medium is addedand DC culture is continued for an additional 46 hours, after whichHUVECs are stimulated with 0.1 ng/mL of IL-1β or 1 ng/mL of TNF-α for aperiod of 2 hours. Total RNA is harvested 48 hours followingtransfection, and real time PCR is performed using primer/probe sets todetect ICAM-1, IRF-1 and SCYB11 in IFN-γ-stimulated cells, or ICAM-1,A20, IL-8 and MIP2α in IL-1β-stimulated and TNF-α-stimulated cells.Expression levels of each gene are normalized to total RNA. Geneexpression levels from cells treated with oligomeric compounds arenormalized to those from untreated control cells. A value greater than100% indicates an induction of the inflammatory response, whereas avalue less than 100% demonstrates a reduction in the inflammatoryresponse.

Oligomeric compounds that inhibit the inflammatory response arecandidate therapeutic compounds with applications in the prevention,treatment or attenuation of conditions associated with hyperstimulationof the immune system, including rheumatoid arthritis, irritable boweldisease, asthma, lupus and multiple sclerosis.

In vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

Mouse Model of Tumorigenesis:

Animal models of tumorigenesis are used in some embodiments of thepresent invention. In this model, tumorigenic cells are injected intoimmunocompromised mice (i.e. nude mice), and subsequent growth of atumor is measured.

Serially transplanted MDA-MB-231 (a human breast carcinoma cell line,American Type Culture Collection, Manassus, Va.) tumors are establishedsubcutaneously in nude mice. Beginning two weeks later, one or more ofthe oligomeric compounds of the invention are administered intravenouslydaily for 14 days at dosages of 15 mg/kg or 30 mg/kg. Control compoundsare also administered at these doses, and a saline control is alsogiven. Tumor growth rates are monitored for the two-week period ofoligonucleotide administration. Activity of the oligomeric compounds ofthe invention is measured by a reduction in tumor growth. Activity ismeasured by reduced tumor volume compared to saline or control compound.Following death or sacrifice of mice, tumor tissue is fixed in 4%formalin, embedded in paraffin, sectioned and stained with hematoxylinand eosin. Tumor tissue sections are evaluated for tumor morphology andsize.

Human A549 lung tumor cells are also injected into nude mouse to producetumors. 200 μl of A549 cells (5×10⁶ cells) are implanted subcutaneouslyin the inner thigh of nude mice. Oligomeric compounds of the inventionare administered twice weekly for four weeks, beginning one weekfollowing tumor cell inoculation. Oligomeric compounds are formulatedwith cationic lipids (LIPOFECTIN™, Invitrogen Corporation, Carlsbad,Calif.) and given subcutaneously in the vicinity of the tumor.Oligomeric compound dosage is 5 mg/kg with 60 mg/kg cationic lipid.Tumor size is recorded weekly. Activity of the oligomeric compounds ofthe invention is measured by reduction in tumor size compared tocontrols.

Xenograft studies are also performed using the U-87 human glioblastomacell line (American Type Culture Collection, Manassus, Va.). Nude miceare injected subcutaneously with 2×10⁷ U-87 cells. Mice are injectedintraperitoneally with one or more of the oligomeric compounds of theinvention or a control compound at dosages of either 15 mg/kg or 30mg/kg for 21 consecutive days beginning 7 days after xenografts areimplanted. Saline-injected animals serve as a control. Tumor volumes aremeasured on days 14, 21, 24, 31 and 35. Activity is measured by reducedtumor volume compared to saline or control compound. Following death orsacrifice of mice, tumor tissue is fixed in 4% formalin, embedded inparaffin, sectioned and stained with hematoxylin and eosin. Tumor tissuesections are evaluated for tumor morphology and size.

Alternatively, intracerebral U-87 xenografts are generated by implantingU-87 glioblastoma cells into the brains of nude mice. Mice are treatedvia continuous intraperitoneal administration with one or more of theoligomeric compounds of the invention at 20 mg/kg, control compound at20 mg/kg or saline beginning on day 7 after xenograft implantation.Activity of the oligomeric compounds of the invention is measured by anincrease in survival time compared to controls. Following death orsacrifice, brain tissue is fixed in 4% formalin, embedded in paraffin,sectioned and stained with hematoxylin and eosin. Brain tissue sectionsare evaluated for tumor growth.

Leptin-Deficient Mice (a Model of Obesity and Diabetes (ob/ob Mice)):

Leptin is a hormone produced by fat cells that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. ob/ob mice have a mutation in the leptin gene which resultsin obesity and hyperglycemia. As such, these mice are a useful model forthe investigation of obesity and diabetes and treatments designed totreat these conditions. ob/ob mice have higher circulating levels ofinsulin and are less hyperglycemic than db/db mice, which harbor amutation in the leptin receptor. In accordance with the presentinvention, the oligomeric compounds of the invention are tested in theob/ob model of obesity and diabetes.

Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) are fed a diet with a fat content of 10-15% and aresubcutaneously injected with the oligomeric compounds of the inventionor a control compound at a dose of 25 mg/kg two times per week for 4weeks. Saline-injected animals, leptin wildtype littermates (i.e. leanlittermates) and ob/ob mice fed a standard rodent diet serve ascontrols. After the treatment period, mice are sacrificed and targetlevels are evaluated in liver, brown adipose tissue (BAT) and whiteadipose tissue (WAT). RNA isolation and target RNA expression levelquantitation are performed as described by other examples herein.

To assess the physiological effects resulting from modulation of target,the ob/ob mice are evaluated at the end of the treatment period forserum lipids, serum free fatty acids, serum cholesterol (CHOL), livertriglycerides, fat tissue triglycerides and liver enzyme levels. Hepaticsteatosis, or clearing of lipids from the liver, is assessed bymeasuring the liver triglyceride content. Hepatic steatosis is assessedby routine histological analysis of frozen liver tissue sections stainedwith oil red O stain, which is commonly used to visualize lipiddeposits, and counterstained with hematoxylin and eosin, to visualizenuclei and cytoplasm, respectively.

The effects of target modulation on glucose and insulin metabolism areevaluated in the ob/ob mice treated with the oligomeric compounds of theinvention. Plasma glucose is measured at the start of the treatment andafter 2 weeks and 4 weeks of treatment. Plasma insulin is similarlymeasured at the beginning of the treatment, and following at 2 weeks andat 4 weeks of treatment. Glucose and insulin tolerance tests are alsoadministered in fed and fasted mice. Mice receive intraperitonealinjections of either glucose or insulin, and the blood glucose andinsulin levels are measured before the insulin or glucose challenge andat 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of ob/ob mice treated with the oligomericcompounds of the invention, the respiratory quotient and oxygenconsumption of the mice are also measured.

The ob/ob mice that receive treatment are evaluated at the end of thetreatment period for the effects of target modulation on the expressionof genes that participate in lipid metabolism, cholesterol biosynthesis,fatty acid oxidation, fatty acid storage, gluconeogenesis and glucosemetabolism. These genes include, but are not limited to, HMG-CoAreductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2,camitine palmitoyltransferase I and glycogen phosphorylase,glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1,lipoprotein lipase and hormone sensitive lipase. mRNA levels in liverand white and brown adipose tissue are quantitated by real-time PCR asdescribed in other examples herein, employing primer/probe sets that aregenerated using published sequences of each gene of interest.

Leptin Receptor-Deficient Mice (a Model of Obesity and Diabetes (db/dbMice)):

db/db mice have a mutation in the leptin receptor gene which results inobesity and hyperglycemia. As such, these mice are a useful model forthe investigation of obesity and diabetes and treatments designed totreat these conditions. db/db mice, which have lower circulating levelsof insulin and are more hyperglycemic than ob/ob mice which harbor amutation in the leptin gene, are often used as a rodent model of type 2diabetes. In accordance with the present invention, oligomeric compoundsof the present invention are tested in the db/db model of obesity anddiabetes.

Seven-week old male C57Bl/6J-Lepr db/db mice (Jackson Laboratory, BarHarbor, Me.) are fed a diet with a fat content of 15-20% and aresubcutaneously injected with one or more of the oligomeric compounds ofthe invention or a control compound at a dose of 25 mg/kg two times perweek for 4 weeks. Saline-injected animals, leptin receptor wildtypelittermates (i.e. lean littermates) and db/db mice fed a standard rodentdiet serve as controls. After the treatment period, mice are sacrificedand target levels are evaluated in liver, BAT and WAT as describedsupra.

To assess the physiological effects resulting from modulation of target,the db/db mice that receive treatment are evaluated at the end of thetreatment period for serum lipids, serum free fatty acids, serumcholesterol (CHOL), liver triglycerides, fat tissue triglycerides andliver enzyme levels. Hepatic steatosis is assessed by measuring theliver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism arealso evaluated in the db/db mice treated with the oligomeric compoundsof the invention. Plasma glucose is measured at the start of thetreatment and after 2 weeks and 4 weeks of treatment. Plasma insulin issimilarly measured at the beginning of the treatment, and following 2weeks and 4 weeks of treatment. Glucose and insulin tolerance tests arealso administered in fed and fasted mice. Mice receive intraperitonealinjections of either glucose or insulin, and the blood glucose levelsare measured before the insulin or glucose challenge and 15, 30, 60, 90and 120 minutes following the injection.

To assess the metabolic rate of db/db mice treated with the oligomericcompounds of the invention, the respiratory quotient and oxygenconsumption of the mice is also measured.

The db/db mice that receive treatment are evaluated at the end of thetreatment period for the effects of target modulation on the expressionof genes that participate in lipid metabolism, cholesterol biosynthesis,fatty acid oxidation, fatty acid storage, gluconeogenesis and glucosemetabolism, as described supra.

Lean Mice on a Standard Rodent Diet:

C57Bl/6 mice are maintained on a standard rodent diet and are used ascontrol (lean) animals. In one embodiment of the present invention, theoligomeric compounds of the invention are tested in normal, leananimals.

Seven-week old male C57Bl/6 mice are fed a diet with a fat content of 4%and are subcutaneously injected with one or more of the oligomericcompounds of the invention or control compounds at a dose of 25 mg/kgtwo times per week for 4 weeks. Saline-injected animals serve as acontrol. After the treatment period, mice are sacrificed and targetlevels are evaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of thetarget, the lean mice that receive treatment are evaluated at the end ofthe treatment period for serum lipids, serum free fatty acids, serumcholesterol (CHOL), liver triglycerides, fat tissue triglycerides andliver enzyme levels. Hepatic steatosis is assessed by measuring theliver triglyceride content and oil red 0 staining, as described supra.

The effects of target modulation on glucose and insulin metabolism arealso evaluated in the lean mice treated with the oligomeric compounds ofthe invention. Plasma glucose is measured at the start of the treatmentand after 2 weeks and 4 weeks of treatment. Plasma insulin is similarlymeasured at the beginning of the treatment, and following 2 weeks and 4weeks of treatment. Glucose and insulin tolerance tests are alsoadministered in fed and fasted mice. Mice receive intraperitonealinjections of either glucose or insulin, and the blood glucose levelsare measured before the insulin or glucose challenge and 15, 30, 60, 90and 120 minutes following the injection.

To assess the metabolic rate of lean mice treated with the oligomericcompounds of the invention, the respiratory quotient and oxygenconsumption of the mice is also measured.

The lean mice that received treatment are evaluated at the end of thetreatment period for the effects of target modulation on the expressionof genes that participate in lipid metabolism, cholesterol biosynthesis,fatty acid oxidation, fatty acid storage, gluconeogenesis and glucosemetabolism, as described supra.

Levin Model of Diet-Induced Obesity in Rats:

The Levin Model is a polygenic model of rats selectively bred to developdiet-induced obesity (DIO) associated with impaired glucose tolerance,dyslipidemia and insulin resistance when fed a high-fat diet (Levin, etal., Am. J. Physiol, 1997, 273, R725-30). The advantage of this model isthat it displays traits more similar to human obesity and glucoseintolerance than in animals that are obese/hyperinsulinemic due togenetic defects e.g. defects in leptin signaling. This model is usefulin investigating the oligomeric compounds of the present invention fortheir ability to affect obesity and related complications, such asimpaired glucose tolerance, dyslipidemia and insulin resistance. Inaccordance with the present invention, the oligomeric compounds of theinvention are tested in the Levin model of diet-induced obesity.

Eight-week old male Levin rats (Charles River Laboratories, Wilmington,Mass.), weighing ˜500 g, are fed a diet with a fat content of 60% foreight weeks, after which they are subcutaneously injected with one ormore of the oligomeric compounds of the invention at a dose of 25mg/kg×2 per week for 8 weeks. Control groups consist of animals injectedwith saline or a control compound and lean littermates fed a standardrodent diet. The control compound is injected at the same dose as thetarget-specific compound.

Throughout the treatment period, the rats are evaluated for foodconsumption, weight gain, as well as serum levels of glucose, insulin,cholesterol, free fatty acids, triglycerides and liver enzymes.

The effects of target modulation on glucose and insulin metabolism arealso evaluated in the Levin rats treated with the oligomeric compoundsof the invention. Plasma glucose and insulin are monitored throughoutthe treatment by analyzing blood samples. Glucose and tolerance areassessed in fed or fasted rats. After blood is collected for baselineglucose and insulin levels, a glucose challenge is administered, afterwhich blood glucose and insulin levels are measured at 15, 20 or 30minute intervals for up to 3 hours. Insulin tolerance is similarlyanalyzed, beginning with blood collection for baseline glucose andinsulin levels, followed by an insulin challenge, after which bloodglucose levels are measured at 15, 20 or 30 minute intervals for up to 3hours. Plasma insulin and glucose are also measured at studytermination.

At the end of the treatment period, the rats are sacrificed. Organs areremoved and weighed, including liver, white adipose tissue, brownadipose tissue and spleen. Target RNA expression levels are measured inall tissues that are isolated, using quantitative real-time PCR. Targetprotein levels are also evaluated by immunoblot analysis usingantibodies that specifically recognize the target protein.

Also evaluated at the end of the treatment period are serum lipids,serum free fatty acids, serum cholesterol (CHOL), liver triglycerides,fat tissue triglycerides and liver enzyme levels. Hepatic steatosis isassessed by measuring the liver triglyceride content and oil red 0staining, as described supra.

The Levin rats that receive treatment are evaluated at the end of thetreatment period for the effects of target modulation on the expressionof genes that participate in lipid metabolism, cholesterol biosynthesis,fatty acid oxidation, fatty acid storage, gluconeogenesis and glucosemetabolism, as described supra.

C57BL/6 on a High-Fat Diet (A Model of Diet-Induced Obesity (DIO)):

The C57BL/6 mouse strain is reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation. Consequently,when these mice are fed a high-fat diet, they develop diet-inducedobesity. Accordingly these mice are a useful model for the investigationof obesity and treatments designed to treat these conditions. In oneembodiment of the present invention, the oligomeric compounds of theinvention are tested in a model of diet-induced obesity.

Male C57BL/6 mice (7-weeks old) receive a 60% fat diet for 8 weeks,after which mice are subcutaneously injected with one or more of theoligomeric compounds of the invention at a dose of 25 mg/kg two timesper week for 4 weeks. Saline-injected or control compound-injectedanimals serve as a control. After the treatment period, mice aresacrificed and target levels are evaluated in liver, BAT and WAT asdescribed supra.

To assess the physiological effects resulting from modulation of target,the diet-induced obese mice that receive treatment are evaluated at theend of the treatment period for serum lipids, serum free fatty acids,serum cholesterol (CHOL), liver triglycerides, fat tissue triglyceridesand liver enzyme levels. Hepatic steatosis is assessed by measuring theliver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism arealso evaluated in the diet-induced obese mice treated with theoligomeric compounds of the invention. Plasma glucose is measured at thestart of treatment and after 2 weeks and 4 weeks of treatment. Plasmainsulin is similarly measured at the beginning of the treatment, andfollowing 2 weeks and 4 weeks of treatment. Glucose and insulintolerance tests are also administered in fed and fasted mice. Micereceive intraperitoneal injections of either glucose or insulin, and theblood glucose and insulin levels are measured before the insulin orglucose challenge and at 15, 20 or 30 minute intervals for up to 3hours.

To assess the metabolic rate of diet-induced obese mice treated with theoligomeric compounds of the invention, the respiratory quotient andoxygen consumption of the mice is also measured.

The diet-induced obese mice that receive treatment are evaluated at theend of the treatment period for the effects of target modulation on theexpression of genes that participate in lipid metabolism, cholesterolbiosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesisand glucose metabolism, as described supra.

P-407 Mouse Model of Hyperlipidemia:

Poloxamer 407 (P-407), an inert block copolymer comprising a hydrophobiccore flanked by hydrophilic polyoxyethelene units has been shown toinduce hyperlipidemia in rodents. In the mouse, one injection,intraperitoneally, of P-407 (0.5 g/kg) produced hypercholesterolemiathat peaked at 24 hours and returned to control levels by 96 hoursfollowing treatment (Palmer, et al., Atherosclerosis, 1998, 136,115-123). Consequently, these mice are a useful model for theinvestigation of compounds that modulate hyperlipidemia. In accordancewith the present invention, the oligomeric compounds of the inventionare tested in the P-407 model of hyperlipidemia.

Seven-week old male C57Bl/6 mice are divided into two groups; (1)control and (2) P-407 injected animals (0.5 g/kg every 3 days, followingan overnight fast). Animals in each group receive either a salineinjection or injection with one or more of the oligomeric compounds ofthe invention or control compounds at 25 mg/kg three times per week or50 mg/kg two times per week. All injections are administeredintraperitoneally.

After the treatment period, mice are sacrificed and target levels areevaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target,the P-407 injected animals that receive treatment are evaluated at theend of the treatment period for serum lipids, serum free fatty acids,serum cholesterol (CHOL), liver triglycerides, fat tissue triglyceridesand liver enzyme levels. Hepatic steatosis is assessed by measuring theliver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism areevaluated in the P-407 injected animals treated with the oligomericcompounds of the invention. Plasma glucose is measured at the start ofthe treatment and after 2 weeks and 4 weeks of treatment. Plasma insulinis similarly measured at the beginning of the treatment, and following 2weeks and 4 weeks of treatment. Glucose and insulin tolerance tests arealso administered in fed and fasted mice. Mice receive intraperitonealinjections of either glucose or insulin, and the blood glucose andinsulin levels are measured before the insulin or glucose challenge andat 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of P-407 injected animals treated with theoligomeric compounds of the invention, the respiratory quotient andoxygen consumption of the mice is measured.

The P-407 injected animals that receive treatment are evaluated at theend of the treatment period for the effects of target modulation on theexpression of genes that participate in lipid metabolism, cholesterolbiosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesisand glucose metabolism, as described supra.

ApoE Knockout Mice (A Model of Dyslipidemia and Obesity):

B6.129P-ApoE^(tm1Unc) knockout mice (herein referred to as ApoE knockoutmice) obtained from The Jackson Laboratory (Bar Harbor, Me.), arehomozygous for the Apoe^(tm1Unc) mutation and show a marked increase intotal plasma cholesterol levels that are unaffected by age or sex. Theseanimals present with fatty streaks in the proximal aorta at 3 months ofage. These lesions increase with age and progress to lesions with lesslipid but more elongated cells, typical of a more advanced stage ofpre-atherosclerotic lesion.

The mutation in these mice resides in the apolipoprotein E (ApoE) gene.The primary role of the ApoE protein is to transport cholesterol andtriglycerides throughout the body. It stabilizes lipoprotein structure,binds to the low density lipoprotein receptor (LDLR) and relatedproteins, and is present in a subclass of HDLs, providing them theability to bind to LDLR. ApoE is expressed most abundantly in the liverand brain. In one embodiment of the present invention, femaleB6.129P-Apoetm1Unc knockout mice (ApoE knockout mice) are used in thefollowing studies to evaluate the oligomeric compounds of the inventionas potential lipid lowering compounds.

Female ApoE knockout mice range in age from 5 to 7 weeks and are placedon a normal diet for 2 weeks before study initiation. ApoE knockout miceare then fed ad libitum a 60% fat diet, with 0.15% added cholesterol toinduce dyslipidemia and obesity. Control animals include ApoE knockoutmice and ApoE wildtype mice (i.e. lean littermates) maintained on ahigh-fat diet with no added cholesterol. After overnight fasting, micefrom each group are dosed intraperitoneally every three days withsaline, 50 mg/kg of a control compound or 5, 25 or 50 mg/kg of one ormore of the oligomeric compounds of the invention.

After the treatment period, mice are sacrificed and target levels areevaluated in liver, BAT and WAT as described supra.

To assess the physiological effects resulting from modulation of target,the ApoE knockout mice that receive treatment are evaluated at the endof the treatment period for serum lipids, serum free fatty acids, serumcholesterol (CHOL), liver triglycerides, fat tissue triglycerides andliver enzyme levels. Hepatic steatosis is assessed by measuring theliver triglyceride content and oil red O staining, as described supra.

The effects of target modulation on glucose and insulin metabolism arealso evaluated in the ApoE knockout mice treated with the oligomericcompounds of the invention. Plasma glucose is measured at the start ofthe treatment and after 2 weeks and 4 weeks of treatment. Plasma insulinis similarly measured at the beginning of the treatment, and following 2weeks and 4 weeks of treatment. Glucose and insulin tolerance tests arealso administered in fed and fasted mice. Mice receive intraperitonealinjections of either glucose or insulin, and the blood glucose andinsulin levels are measured before the insulin or glucose challenge andat 15, 20 or 30 minute intervals for up to 3 hours.

To assess the metabolic rate of ApoE knockout mice treated with theoligomeric compounds of the invention, the respiratory quotient andoxygen consumption of the mice are measured.

The ApoE knockout mice that receive treatment are evaluated at the endof the treatment period for the effects of target modulation on theexpression of genes that participate in lipid metabolism, cholesterolbiosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesisand glucose metabolism, as described supra.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner. Throughout theseexamples, molecular cloning reactions, and other standard recombinantDNA techniques, were carried out according to methods described inManiatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., ColdSpring Harbor Press (1989), using commercially available reagents,except where otherwise noted.

EXAMPLES Example 1 Oligomeric Compounds Targeting Small Non-Coding RNAs

In accordance with the present invention, a series of oligomericcompounds are designed to target different regions of small non-codingtarget RNAs. The oligomeric compounds can be investigated for theireffect on small non-coding RNA levels by quantitative real-time PCR. Thetarget regions to which these sequences are complementary are hereinreferred to as “suitable target regions”.

Example 2 Oligomeric Compounds that Mimic or Replace Small Non-CodingRNAs

In accordance with the present invention, a series of oligomericcompounds are designed to mimic the structure and/or function of smallnon-coding RNAs. These mimics may include isolated single-, double-, ormultiple-stranded compounds, any of which may include regions ofintrastrand nucleobase complementarity, said regions capable of foldingand forming a molecule with fully or partially double-stranded ormultiple-stranded character based on regions of precise or imperfectcomplementarity. The oligomeric compound mimics can then be investigatedfor their effects on a cell, tissue or organism system lackingendogenous small non-coding RNAs or systems with aberrant expression ofsmall non-coding RNAs using the screening methods disclosed herein orthose commonly used in the art. Changes in levels, expression orfunction of the small non-coding RNA or its downstream target nucleicacid levels can be analyzed by quantitative real-time PCR as described,supra.

Example 3 Pri-miRNAs Targeted by Compounds of the Present Invention

In accordance with the present invention, oligomeric compounds weredesigned to target one or more microRNA (miRNA) genes or gene products.Certain pri-miRNAs have been reported by Lim et al. Science, 2003, 299;1540; in Brevia (detailed in the supplemental online materials;www.sciencemag.org/cgi/content/full/299/5612/1540/DC1) and these wereused as starting targets.

A list of pri-miRNAs targeted is shown in Table 1. The gene name foreach of the 188 targets (assigned by Lim et al.) is given in the table.For those pri-miRNAs that did not produce an identifiable miRNAdetectable by PCR in the Lim publication, the position and sequence ofthe miRNAs were identified herein and are referred to as novel orhypothetical miRNAs. Also shown is the Genbank Accession number of thesource sequence from which the pri-miRNA was extracted. The sequence isset forth in the Sequence Listing and is written in the 5′ to 3′direction and is represented in the DNA form. It is understood that aperson having ordinary skill in the art would be able to convert thesequence of the targets to their RNA form by simply replacing thethymidine (T) with uracil (U) in the sequence.

TABLE 1 pri-miRNAs Genbank Accession # of source SEQ pri-miRNA sequenceID NO mir-140 NT_037896.1 4 mir-30a NT_007299.11 5 mir-34 NT_028054.10 6mir-29b-1 NT_021877.13 7 mir-29b-2 NT_007933.10 8 mir-16-3 NT_005612.119 mir-203 NT_026437.9 10 mir-7-1 NT_023935.13 11 mir-10b NT_037537.1 12mir-128a NT_034487.2 13 mir-153-1 NT_005403.11 14 mir-153-2 NT_007741.1015 hypothetical miRNA-013 NT_010194.13 16 mir-27b NT_008476.13 17 mir-96NT_007933.10 18 mir-17as/mir-91 NT_009952.11 19 mir-123/mir-126asNT_024000.13 20 mir-132 NT_010692.9 21 mir-108-1 NT_010799.11 22 mir-23bNT_008476.13 23 let-7i NT_009711.13 24 mir-212 NT_010692.9 25hypothetical miRNA-023 NT_004658.12 26 mir-131-2 NT_029973.6 27 let-7bNT_011523.8 28 mir-1d NT_035608.1 29 mir-122a NT_033907.3 30 mir-22NT_010692.9 31 mir-92-1 NT_009952.11 32 hypothetical miRNA-030NT_007933.10 33 mir-142 NT_010783.11 34 mir-183 NT_007933.10 35hypothetical miRNA-033 NT_011588.11 36 mir-214 NT_029874.7 37 mir-143NT_006859.11 38 mir-192-1 NT_033241.3 39 mir-192-2 NT_033241.3 39mir-192-3 NT_033241.3 39 hypothetical miRNA-039 NT_028392.4 42hypothetical miRNA-040 NT_023148.9 43 hypothetical miRNA-041NT_023089.11 44 let-7a-3 NT_011523.8 45 hypothetical miRNA-043NT_004902.12 46 hypothetical miRNA-044 NT_009952.11 47 mir-181aNT_017568.11 48 let-7a-1 NT_008476.13 49 mir-205 NT_021877.13 50mir-103-1 NT_037665.1 51 mir-26a NT_005580.13 52 mir-33a NT_011520.8 53mir-196-2 NT_009458.12 54 mir-107 NT_033890.3 55 mir-106 NT_011786.11 56let-7f-1 NT_008476.13 57 hypothetical miRNA-055 NT_006713.11 58 mir-29cNT_021877.13 59 mir-130a NT_033903.3 60 hypothetical miRNA-058NT_037537.1 61 mir-218-1 NT_006316.13 62 mir-124a-2 NT_008183.13 63mir-21 NT_035426.2 64 mir-16-1 NT_033922.3 65 mir-144 NT_010799.11 66mir-221 NT_011568.10 67 mir-222 NT_011568.10 68 mir-30d NT_028251.8 69mir-19b-2 NT_011786.11 70 mir-128b NT_005580.13 71 hypotheticalmiRNA-069 NT_017568.11 72 hypothetical miRNA-070 NT_005375.11 73hypothetical miRNA-071 NT_011512.7 74 mir-29b-3 NT_007933.10 75mir-129-2 NT_009237.13 76 mir-133b NT_007592.11 77 hypotheticalmiRNA-075 NT_006044.8 78 let-7d NT_008476.13 79 mir-15b NT_005612.11 80mir-29a-1 NT_007933.10 81 hypothetical miRNA-079 NT_021907.13 82mir-199b NT_017568.11 83 mir-129-1 NT_007933.10 84 let-7e NT_011109.1385 hypothetical miRNA-083 NT_024524.11 86 let-7c NT_011512.7 87 mir-204NT_008580.11 88 mir-145 NT_006859.11 89 mir-124a-1 NT_019483.13 90hypothetical miRNA-088 NT_011519.9 91 mir-213 NT_029862.8 92hypothetical miRNA-090 NT_006171.13 93 mir-20 NT_009952.11 94 mir-133a-1NT_011044.11 95 mir-138-2 NT_010498.11 96 mir-98 NT_011799.10 97mir-196-1 NT_010783.11 98 mir-125b-1 NT_033899.3 99 mir-199a-2NT_029874.7 100 mir-29a-2 NT_007933.10 101 hypothetical miRNA-099NT_016297.12 102 mir-181b NT_029862.8 103 hypothetical miRNA-101NT_030828.7 104 mir-141 NT_035206.1 105 mir-131-1 NT_004858.13 106mir-133a-2 NT_035608.1 107 hypothetical miRNA-105 NT_017795.13 108hypothetical miRNA-106 NT_017795.13 109 hypothetical miRNA-107NT_008583.13 110 mir-1b NT_011044.11 111 mir-18 NT_009952.11 112 mir-220NT_011588.11 113 hypothetical miRNA-111 NT_004525.13 114 mir-7-3NT_011255.11 115 mir-218-2 NT_023132.10 116 mir-24-2 NT_031915.4 117mir-24-1 NT_008476.13 118 mir-103-2 NT_011387.8 119 mir-211 NT_010363.13120 mir-101-3 NT_008413.13 121 mir-30b NT_028251.8 122 hypotheticalmiRNA-120 NT_009952.11 123 let-7a-4 NT_033899.3 124 mir-10a NT_010783.11125 mir-19a NT_009952.11 126 let-7f-2 NT_011799.10 127 mir-15a-1NT_010393.11 128 mir-108-2 NT_034392.2 129 mir-137 NT_033951.3 130mir-219 NT_007592.11 131 mir-148b NT_009458.12 132 mir-130b NT_011520.8133 mir-19b-1 NT_009952.11 134 let-7a-2 NT_033899.3 135 mir-216NT_005375.11 136 mir-100-1 NT_033899.3 137 mir-100-2 NT_033899.3 137mir-187 NT_010966.11 139 hypothetical miRNA-137 NT_011387.8 140hypothetical miRNA-138 NT_008902.13 141 hypothetical miRNA-139NT_008902.13 142 mir-124a-3 NT_011333.5 143 mir-7-2 NT_033276.3 144hypothetical miRNA-142 NT_033317.3 145 hypothetical miRNA-143NT_007819.11 146 hypothetical miRNA-144 NT_010783.11 147 mir-210NT_035113.2 148 mir-215 NT_021953.13 149 mir-223 NT_011669.11 150mir-131-3 NT_033276.3 151 mir-199a-1 NT_011176.13 152 mir-30cNT_007299.11 153 mir-101-1 NT_029865.8 154 mir-101-2 NT_029865.8 154hypothetical miRNA-153 NT_005332.11 156 hypothetical miRNA-154NT_030828.7 157 mir-26b NT_005403.11 158 hypothetical miRNA-156NT_029289.7 159 mir-152 NT_010783.11 160 mir-135-1 NT_005986.13 161mir-135-2 NT_009681.13 162 mir-217 NT_005375.11 163 hypotheticalmiRNA-161 NT_004658.12 164 mir-15a-2 NT_033922.3 165 let-7g NT_005986.13166 hypothetical miRNA-164 NT_010783.11 167 mir-33b NT_030843.4 168hypothetical miRNA-166 NT_011588.11 169 mir-16-2 NT_033922.3 170hypothetical miRNA-168 NT_011520.8 171 hypothetical miRNA-169NT_007933.10 172 hypothetical miRNA-170 NT_005151.11 173 hypotheticalmiRNA-171 NT_006171.13 174 hypothetical miRNA-172 NT_037752.1 175hypothetical miRNA-173 NT_008413.13 176 mir-182 NT_007933.10 177hypothetical miRNA-175 NT_006258.12 178 hypothetical miRNA-176NT_025004.11 179 hypothetical miRNA-177 NT_023098.7 180 hypotheticalmiRNA-178 NT_037537.1 181 hypothetical miRNA-179 NT_010194.13 182hypothetical miRNA-180 NT_010363.13 183 hypothetical miRNA-181NT_033899.3 184 mir-148a NT_007819.11 185 hypothetical miRNA-183NT_010363.13 186 mir-23a NT_031915.4 187 hypothetical miRNA-185NT_007592.11 188 hypothetical miRNA-186 NT_008705.13 189 mir-181cNT_031915.4 190 hypothetical miRNA-188 NT_023148.9 191

Example 4 miRNAs Within pri-miRNAs

miRNAs found within the pri-miRNA structures disclosed above were usedin certain embodiments of the present invention. These miRNAs representtarget nucleic acids to which the oligomeric compounds of the presentinvention were designed. The oligomeric compounds of the presentinvention can also be designed to mimic the miRNA while incorporatingcertain chemical modifications that alter one or more properties of themimic, thereby creating a construct with superior properties over theendogenous miRNA. The miRNA target sequences are shown in Table 2.

TABLE 2 miRNAs found within pri-miRNAs miRNA sequence (DNA form; whereSEQ ID Pri-miRNA T replaces U in RNA) NO mir-140 AGTGGTTTTACCCTATGGTAG192 mir-30a CTTTCAGTCGGATGTTTGCAGC 193 mir-34 TGGCAGTGTCTTAGCTGGTTGT 194mir-29b-1 TAGCACCATTTGAAATCAGTGTT 195 mir-29b-2 TAGCACCATTTGAAATCAGTGTT195 mir-16-3 TAGCAGCACGTAAATATTGGCG 196 mir-203 GTGAAATGTTTAGGACCACTAG197 mir-7-1 TGGAAGACTAGTGATTTTGTT 198 mir-10b TACCCTGTAGAACCGAATTTGT 199mir-128a TCACAGTGAACCGGTCTCTTTT 200 mir-153-1 TTGCATAGTCACAAAAGTGA 201mir-153-2 TTGCATAGTCACAAAAGTGA 201 mir-27b TTCACAGTGGCTAAGTTCTG 202mir-96 TTTGGCACTAGCACATTTTTGC 203 mir-17as/mir-91CAAAGTGCTTACAGTGCAGGTAGT 204 mir-123/mir-126as CATTATTACTTTTGGTACGCG 205mir-132 TAACAGTCTACAGCCATGGTCGC 206 mir-108-1 ATAAGGATTTTTAGGGGCATT 207mir-23b ATCACATTGCCAGGGATTACCAC 208 let-7i TGAGGTAGTAGTTTGTGCT 209mir-212 TAACAGTCTCCAGTCACGGCC 210 mir-131-2 TAAAGCTAGATAACCGAAAGT 211let-7b TGAGGTAGTAGGTTGTGTGGTT 212 mir-1d TGGAATGTAAAGAAGTATGTAT 213mir-122a TGGAGTGTGACAATGGTGTTTGT 214 mir-22 AAGCTGCCAGTTGAAGAACTGT 215mir-92-1 TATTGCACTTGTCCCGGCCTGT 216 mir-142 CATAAAGTAGAAAGCACTAC 217mir-183 TATGGCACTGGTAGAATTCACTG 218 mir-214 ACAGCAGGCACAGACAGGCAG 219mir-143 TGAGATGAAGCACTGTAGCTCA 220 mir-192-1 CTGACCTATGAATTGACAGCC 221mir-192-2 CTGACCTATGAATTGACAGCC 221 mir-192-3 CTGACCTATGAATTGACAGCC 221let-7a-3 TGAGGTAGTAGGTTGTATAGTT 222 mir-181a AACATTCAACGCTGTCGGTGAGT 223let-7a-1 TGAGGTAGTAGGTTGTATAGTT 222 mir-205 TCCTTCATTCCACCGGAGTCTG 224mir-103-1 AGCAGCATTGTACAGGGCTATGA 225 mir-26a TTCAAGTAATCCAGGATAGGCT 226mir-33a GTGCATTGTAGTTGCATTG 227 mir-196-2 TAGGTAGTTTCATGTTGTTGGG 228mir-107 AGCAGCATTGTACAGGGCTATCA 229 mir-106 AAAAGTGCTTACAGTGCAGGTAGC 230let-7f-1 TGAGGTAGTAGATTGTATAGTT 231 mir-29c CTAGCACCATTTGAAATCGGTT 232mir-130a CAGTGCAATGTTAAAAGGGC 233 mir-218-1 TTGTGCTTGATCTAACCATGT 234mir-124a-2 TTAAGGCACGCGGTGAATGCCA 235 mir-21 TAGCTTATCAGACTGATGTTGA 236mir-16-1 TAGCAGCACGTAAATATTGGCG 196 mir-144 TACAGTATAGATGATGTACTAG 237mir-221 AGCTACATTGTCTGCTGGGTTTC 238 mir-222 AGCTACATCTGGCTACTGGGTCTC 239mir-30d TGTAAACATCCCCGACTGGAAG 240 mir-19b-2 TGTGCAAATCCATGCAAAACTGA 241mir-128b TCACAGTGAACCGGTCTCTTTC 242 mir-29b-3 TAGCACCATTTGAAATCAGTGTT195 mir-129-2 CTTTTTGCGGTCTGGGCTTGC 243 mir-133b TTGGTCCCCTTCAACCAGCTA244 let-7d AGAGGTAGTAGGTTGCATAGT 245 mir-15b TAGCAGCACATCATGGTTTACA 246mir-29a-1 CTAGCACCATCTGAAATCGGTT 247 mir-199b CCCAGTGTTTAGACTATCTGTTC248 mir-129-1 CTTTTTGCGGTCTGGGCTTGC 243 let-7e TGAGGTAGGAGGTTGTATAGT 249let-7c TGAGGTAGTAGGTTGTATGGTT 250 mir-204 TTCCCTTTGTCATCCTATGCCT 251mir-145 GTCCAGTTTTCCCAGGAATCCCTT 252 mir-124a-1 TTAAGGCACGCGGTGAATGCCA235 mir-213 ACCATCGACCGTTGATTGTACC 253 mir-20 TAAAGTGCTTATAGTGCAGGTAG254 mir-133a-1 TTGGTCCCCTTCAACCAGCTGT 255 mir-138-2 AGCTGGTGTTGTGAATC256 mir-98 TGAGGTAGTAAGTTGTATTGTT 257 mir-196-1 TAGGTAGTTTCATGTTGTTGGG228 mir-125b-1 TCCCTGAGACCCTAACTTGTGA 258 mir-199a-2CCCAGTGTTCAGACTACCTGTTC 259 mir-29a-2 CTAGCACCATCTGAAATCGGTT 247mir-181b AACATTCATTGCTGTCGGTGGGTT 260 mir-141 AACACTGTCTGGTAAAGATGG 261mir-131-1 TAAAGCTAGATAACCGAAAGT 211 mir-133a-2 TTGGTCCCCTTCAACCAGCTGT255 mir-1b TGGAATGTAAAGAAGTATGTAT 213 mir-18 TAAGGTGCATCTAGTGCAGATA 262mir-220 CCACACCGTATCTGACACTTT 263 mir-7-3 TGGAAGACTAGTGATTTTGTT 198mir-218-2 TTGTGCTTGATCTAACCATGT 234 mir-24-2 TGGCTCAGTTCAGCAGGAACAG 264mir-24-1 TGGCTCAGTTCAGCAGGAACAG 264 mir-103-2 AGCAGCATTGTACAGGGCTATGA225 mir-211 TTCCCTTTGTCATCCTTCGCCT 264 mir-101-3 TACAGTACTGTGATAACTGA265 mir-30b TGTAAACATCCTACACTCAGC 266 let-7a-4 TGAGGTAGTAGGTTGTATAGTT222 mir-10a TACCCTGTAGATCCGAATTTGTG 267 mir-19a TGTGCAAATCTATGCAAAACTGA268 let-7f-2 TGAGGTAGTAGATTGTATAGTT 231 mir-15a-1 TAGCAGCACATAATGGTTTGTG269 mir-108-2 ATAAGGATTTTTAGGGGCATT 207 mir-137 TATTGCTTAAGAATACGCGTAG270 mir-219 TGATTGTCCAAACGCAATTCT 271 mir-148b TCAGTGCATCACAGAACTTTGT272 mir-130b CAGTGCAATGATGAAAGGGC 273 mir-19b-1 TGTGCAAATCCATGCAAAACTGA241 let-7a-2 TGAGGTAGTAGGTTGTATAGTT 222 mir-216 TAATCTCAGCTGGCAACTGTG274 mir-100-1 AACCCGTAGATCCGAACTTGTG 275 mir-100-2AACCCGTAGATCCGAACTTGTG 275 mir-187 TCGTGTCTTGTGTTGCAGCCGG 276 mir-124a-3TTAAGGCACGCGGTGAATGCCA 235 mir-7-2 TGGAAGACTAGTGATTTTGTT 198 mir-210CTGTGCGTGTGACAGCGGCTG 277 mir-215 ATGACCTATGAATTGACAGAC 278 mir-223TGTCAGTTTGTCAAATACCCC 279 mir-131-3 TAAAGCTAGATAACCGAAAGT 211 mir-199a-1CCCAGTGTTCAGACTACCTGTTC 259 mir-30c TGTAAACATCCTACACTCTCAGC 280mir-101-1 TACAGTACTGTGATAACTGA 265 mir-101-2 TACAGTACTGTGATAACTGA 265mir-26b TTCAAGTAATTCAGGATAGGTT 281 mir-152 TCAGTGCATGACAGAACTTGG 282mir-135-1 TATGGCTTTTTATTCCTATGTGAT 283 mir-135-2TATGGCTTTTTATTCCTATGTGAT 283 mir-217 TACTGCATCAGGAACTGATTGGAT 284mir-15a-2 TAGCAGCACATAATGGTTTGTG 269 let-7g TGAGGTAGTAGTTTGTACAGT 285mir-33b GTGCATTGCTGTTGCATTG 286 mir-16-2 TAGCAGCACGTAAATATTGGCG 196mir-182 TTTGGCAATGGTAGAACTCACA 287 mir-148a TCAGTGCACTACAGAACTTTGT 288mir-23a ATCACATTGCCAGGGATTTCC 289 mir-181c AACATTCAACCTGTCGGTGAGT 290

Example 5 Uniform 2′-MOE Phosphorothioate (PS) Oligomeric CompoundsTargeting miRNAs

In accordance with the present invention, a series of oligomericcompounds were designed and synthesized to target miRNA sequencesdisclosed by Lim et al. Science, 2003, 299, 1540. The compounds areshown in Table 3. “Pri-miRNA” indicates the particular pri-miRNA whichcontains the miRNA that the oligomeric compound was designed to target.All compounds in Table 3 are composed of 2′-methoxyethoxy (2′-MOE)nucleotides throughout and the internucleoside (backbone) linkages arephosphorothioate (P═S) throughout. All cytidine residues are5-methylcytidines. The compounds can be analyzed for their effect onmiRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR asdescribed, supra, or they can be used in other assays to investigate therole of miRNAs or the function of targets downstream of miRNAs.

TABLE 3 Uniform 2′-MOE PS Compounds targeting miRNAs ISIS SEQ Number IDNO Sequence Pri-miRNA 327873 291 CTACCATAGGGTAAAACCACT mir-140 327874292 GCTGCAAACATCCGACTGAAAG mir-30a 327875 293 ACAACCAGCTAAGACACTGCCAmir-34 327876 294 AACACTGATTTCAAATGGTGCTA mir-29b-1 327877 295CGCCAATATTTACGTGCTGCTA mir-16-3 327878 296 CTAGTGGTCCTAAACATTTCACmir-203 327879 297 AACAAAATCACTAGTCTTCCA mir-7-1 327880 298ACAAATTCGGTTCTACAGGGTA mir-10b 327881 299 AAAAGAGACCGGTTCACTGTGAmir-128a 327882 300 TCACTTTTGTGACTATGCAA mir-153-1 327883 301CAGAACTTAGCCACTGTGAA mir-27b 327884 302 GCAAAAATGTGCTAGTGCCAAA mir-96327885 303 ACTACCTGCACTGTAAGCACTTTG mir-17as/ mir-91 327886 304CGCGTACCAAAAGTAATAATG mir-123/mir- 126as 327887 305GCGACCATGGCTGTAGACTGTTA mir-132 327888 306 AATGCCCCTAAAAATCCTTATmir-108-1 327889 307 GTGGTAATCCCTGGCAATGTGAT mir-23b 327890 308AGCACAAACTACTACCTCA let-7i 327891 309 GGCCGTGACTGGAGACTGTTA mir-212327892 310 ACTTTCGGTTATCTAGCTTTA mir-131-2/ mir-9 327893 311AACCACACAACCTACTACCTCA let-7b 327894 312 ATACATACTTCTTTACATTCCA mir-1d327895 313 ACAAACACCATTGTCACACTCCA mir-122a 327896 314ACAGTTCTTCAACTGGCAGCTT mir-22 327897 315 ACAGGCCGGGACAAGTGCAATA mir-92-1327898 316 GTAGTGCTTTCTACTTTATG mir-142 327899 317CAGTGAATTCTACCAGTGCCATA mir-183 327900 318 CTGCCTGTCTGTGCCTGCTGT mir-214327901 319 TGAGCTACAGTGCTTCATCTCA mir-143 327902 320GGCTGTCAATTCATAGGTCAG mir-192-1 327903 321 AACTATACAACCTACTACCTCAlet-7a-3 327904 322 ACTCACCGACAGCGTTGAATGTT mir-181a 327905 323CAGACTCCGGTGGAATGAAGGA mir-205 327906 324 TCATAGCCCTGTACAATGCTGCTmir-103-1 327907 325 AGCCTATCCTGGATTACTTGAA mir-26a 327908 326CAATGCAACTACAATGCAC mir-33a 327909 327 CCCAACAACATGAAACTACCTA mir-196-2327910 328 TGATAGCCCTGTACAATGCTGCT mir-107 327911 329GCTACCTGCACTGTAAGCACTTTT mir-106 327912 330 AACTATACAATCTACTACCTCAlet-7f-1 327913 331 AACCGATTTCAAATGGTGCTAG mir-29c 327914 332GCCCTTTTAACATTGCACTG mir-130a 327915 333 ACATGGTTAGATCAAGCACAA mir-218-1327916 334 TGGCATTCACCGCGTGCCTTAA mir-124a-2 327917 335TCAACATCAGTCTGATAAGCTA mir-21 327918 336 CTAGTACATCATCTATACTGTA mir-144327919 337 GAAACCCAGCAGACAATGTAGCT mir-221 327920 338GAGACCCAGTAGCCAGATGTAGCT mir-222 327921 339 CTTCCAGTCGGGGATGTTTACAmir-30d 327922 340 TCAGTTTTGCATGGATTTGCACA mir-19b-2 327923 341GAAAGAGACCGGTTCACTGTGA mir-128b 327924 342 GCAAGCCCAGACCGCAAAAAGmir-129-2 327925 343 TAGCTGGTTGAAGGGGACCAA mir-133b 327926 344ACTATGCAACCTACTACCTCT let-7d 327927 345 TGTAAACCATGATGTGCTGCTA mir-15b327928 346 AACCGATTTCAGATGGTGCTAG mir-29a-1 327929 347GAACAGATAGTCTAAACACTGGG mir-199b 327930 348 ACTATACAACCTCCTACCTCA let-7e327931 349 AACCATACAACCTACTACCTCA let-7c 327932 350AGGCATAGGATGACAAAGGGAA mir-204 327933 351 AAGGGATTCCTGGGAAAACTGGACmir-145 327934 352 GGTACAATCAACGGTCGATGGT mir-213 327935 353CTACCTGCACTATAAGCACTTTA mir-20 327936 354 ACAGCTGGTTGAAGGGGACCAAmir-133a-1 327937 355 GATTCACAACACCAGCT mir-138-2 327938 356AACAATACAACTTACTACCTCA mir-98 327939 357 TCACAAGTTAGGGTCTCAGGGAmir-125b-1 327940 358 GAACAGGTAGTCTGAACACTGGG mir-199a-2 327941 359AACCCACCGACAGCAATGAATGTT mir-181b 327942 360 CCATCTTTACCAGACAGTGTTmir-141 327943 361 TATCTGCACTAGATGCACCTTA mir-18 327944 362AAAGTGTCAGATACGGTGTGG mir-220 327945 363 CTGTTCCTGCTGAACTGAGCCA mir-24-2327946 364 AGGCGAAGGATGACAAAGGGAA mir-211 327947 365TCAGTTATCACAGTACTGTA mir-101-3 327948 366 GCTGAGTGTAGGATGTTTACA mir-30b327949 367 CACAAATTCGGATCTACAGGGTA mir-10a 327950 368TCAGTTTTGCATAGATTTGCACA mir-19a 327951 369 CACAAACCATTATGTGCTGCTAmir-15a-1 327952 370 CTACGCGTATTCTTAAGCAATA mir-137 327953 371AGAATTGCGTTTGGACAATCA mir-219 327954 372 ACAAAGTTCTGTGATGCACTGA mir-148b327955 373 GCCCTTTCATCATTGCACTG mir-130b 327956 374CACAGTTGCCAGCTGAGATTA mir-216 327957 375 CACAAGTTCGGATCTACGGGTTmir-100-1 327958 376 CCGGCTGCAACACAAGACACGA mir-187 327959 377CAGCCGCTGTCACACGCACAG mir-210 327960 378 GTCTGTCAATTCATAGGTCAT mir-215327961 379 GGGGTATTTGACAAACTGACA mir-223 327962 380GCTGAGAGTGTAGGATGTTTACA mir-30c 327963 381 AACCTATCCTGAATTACTTGAAmir-26b 327964 382 CCAAGTTCTGTCATGCACTGA mir-152 327965 383ATCACATAGGAATAAAAAGCCATA mir-135-1 327966 384 ATCCAATCAGTTCCTGATGCAGTAmir-217 327967 385 ACTGTACAAACTACTACCTCA let-7g 327968 386CAATGCAACAGCAATGCAC mir-33b 327969 387 TGTGAGTTCTACCATTGCCAAA mir-182327970 388 ACAAAGTTCTGTAGTGCACTGA mir-148a 327971 389GGAAATCCCTGGCAATGTGAT mir-23a 327972 390 ACTCACCGACAGGTTGAATGTT mir-181c

Example 6 Uniform 2′-MOE Phosphorothioate (PS) Oligomeric CompoundsTargeting Novel miRNAs

In accordance with the present invention, a series of oligomericcompounds were designed and synthesized to target novel miRNAs. Thecompounds are shown in Table 4. “Pri-miRNA” indicates the particularpri-miRNA defined herein which contains the miRNA that the oligomericcompound was designed to target. The sequence of the compounds representthe full complement of the novel miRNA defined herein. All compounds inTable 4 are composed of 2′-methoxyethoxy (2′-MOE) nucleotides throughoutand the internucleoside (backbone) linkages are phosphorothioate (P═S)throughout. All cytidine residues are 5-methylcytidines. The compoundscan be analyzed for their effect miRNA, pre-miRNA or pri-miRNA levels byquantitative real-time PCR as described, supra, or they can be used inother assays to investigate the role of miRNAs or downstream targets ofmiRNAs.

TABLE 4 Uniform 2′-MOE PS Compounds targeting novel pri-miRNAs ISISSequence Number SEQ ID NO (5′-3′) Pri-miRNA 328089 391ACTGTAGGAATATGTTTGATA hypothetical miRNA-013 328090 392ATTAAAAAGTCCTCTTGCCCA hypothetical miRNA-023 328091 393GCTGCCGTATATGTGATGTCA hypothetical miRNA-030 328092 394GGTAGGTGGAATACTATAACA hypothetical miRNA-033 328093 395TAAACATCACTGCAAGTCTTA hypothetical miRNA-039 328094 396TTGTAAGCAGTTTTGTTGACA hypothetical miRNA-040 328095 397TCACAGAGAAAACAACTGGTA hypothetical miRNA-041 328096 398CCTCTCAAAGATTTCCTGTCA hypothetical miRNA-043 328097 399TGTCAGATAAACAGAGTGGAA hypothetical miRNA-044 328098 400GAGAATCAATAGGGCATGCAA hypothetical miRNA-055 328099 401AAGAACATTAAGCATCTGACA hypothetical miRNA-058 328100 402AATCTCTGCAGGCAAATGTGA hypothetical miRNA-070 328101 403AAACCCCTATCACGATTAGCA hypothetical miRNA-071 328102 404GCCCCATTAATATTTTAACCA hypothetical miRNA-075 328103 405CCCAATATCAAACATATCA hypothetical miRNA-079 328104 406TATGATAGCTTCCCCATGTAA hypothetical miRNA-083 328105 407CCTCAATTATTGGAAATCACA hypothetical miRNA-088 328106 408ATTGATGCGCCATTTGGCCTA hypothetical miRNA-090 328107 409CTGTGACTTCTCTATCTGCCT hypothetical miRNA-099 328108 410AAACTTGTTAATTGACTGTCA hypothetical miRNA-101 328109 411AAAGAAGTATATGCATAGGAA hypothetical miRNA-105 328110 412GATAAAGCCAATAAACTGTCA hypothetical miRNA-107 328111 413TCCGAGTCGGAGGAGGAGGAA hypothetical miRNA-111 328112 414ATCATTACTGGATTGCTGTAA hypothetical miRNA-120 328113 415CAAAAATTATCAGCCAGTTTA hypothetical miRNA-137 328114 416AATCTCATTTTCATACTTGCA hypothetical miRNA-138 328115 417AGAAGGTGGGGAGCAGCGTCA hypothetical miRNA-142 328116 418CAAAATTGCAAGCAAATTGCA hypothetical miRNA-143 328117 419TCCACAAAGCTGAACATGTCT hypothetical miRNA-144 328118 420TATTATCAGCATCTGCTTGCA hypothetical miRNA-153 328119 421AATAACACACATCCACTTTAA hypothetical miRNA-154 328120 422AAGAAGGAAGGAGGGAAAGCA hypothetical miRNA-156 328121 423ATGACTACAAGTTTATGGCCA hypothetical miRNA-161 328122 424CAAAACATAAAAATCCTTGCA hypothetical miRNA-164 328123 425TTACAGGTGCTGCAACTGGAA hypothetical miRNA-166 328124 426AGCAGGTGAAGGCACCTGGCT hypothetical miRNA-168 328125 427TATGAAATGCCAGAGCTGCCA hypothetical miRNA-169 328126 428CCAAGTGTTAGAGCAAGATCA hypothetical miRNA-170 328127 429AACGATAAAACATACTTGTCA hypothetical miRNA-171 328128 430AGTAACTTCTTGCAGTTGGA hypothetical miRNA-172 328129 431AGCCTCCTTCTTCTCGTACTA hypothetical miRNA-173 328130 432ACCTCAGGTGGTTGAAGGAGA hypothetical miRNA-175 328131 433ATATGTCATATCAAACTCCTA hypothetical miRNA-176 328132 434GTGAGAGTAGCATGTTTGTCT hypothetical miRNA-177 328133 435TGAAGGTTCGGAGATAGGCTA hypothetical miRNA-178 328134 436AATTGGACAAAGTGCCTTTCA hypothetical miRNA-179 328135 437ACCGAACAAAGTCTGACAGGA hypothetical miRNA-180 328136 438AACTACTTCCAGAGCAGGTGA hypothetical miRNA-181 328137 439GTAAGCGCAGCTCCACAGGCT hypothetical miRNA-183 328138 440GAGCTGCTCAGCTGGCCATCA hypothetical miRNA-185 328139 441TACTTTTCATTCCCCTCACCA hypothetical miRNA-188

Example 7 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and aDeoxy Gap Targeting pri-miRNAs

In accordance with the present invention, a series of oligomericcompounds were designed and synthesized to target different regions ofpri-miRNA structures. The compounds are shown in Table 5. “Pri-miRNA”indicates the particular pri-miRNA which contains the miRNA that theoligomeric compound was designed to target. All compounds in Table 5 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines. The compounds can be analyzed for theireffect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-timePCR as described, supra, or they can be used in other assays toinvestigate the role of miRNAs or miRNA downstream targets.

TABLE 5 Chimeric phosphorothioate oligomeric compounds having 2′-MOEwings and a deoxy gap targeting pri-miRNAs ISIS Number SEQ ID NOSequence pri-miRNA 328333 442 AGAACAGCATGACGTAACCT mir-140 328334 443GCCCATCTGTGGCTTCACAG mir-30a 328335 444 GAAGTCCGAGGCAGTAGGCA mir-30a328336 445 CTTCCTTACTATTGCTCACA mir-34 328337 446 GCTAGATACAAAGATGGAAAmir-29b-1 328338 447 CTAGACAATCACTATTTAAA mir-29b-2 328339 448GCAGCGCAGCTGGTCTCCCC mir-29b-2 328340 449 TAATATATATTTCACTACGC mir-16-3328341 450 TGCTGTATCCCTGTCACACT mir-16-3 328342 451 CAATTGCGCTACAGAACTGTmir-203 328343 452 TCGATTTAGTTATCTAAAAA mir-7-1 328344 453CTGTAGAGGCATGGCCTGTG mir-7-1 328345 454 TGACTATACGGATACCACAC mir-10b328346 455 GGAACAAGGCCAATTATTGC mir-128a 328347 456 AGAAATGTAAACCTCTCAGAmir-128a 328348 457 AGCTGTGAGGGAGAGAGAGA mir-153-1 328349 458CTGGAGTGAGAATACTAGCT mir-153-1 328350 459 ACTGGGCTCATATTACTAGC mir-153-2328351 460 TTGGATTAAATAACAACCTA hypothetical miRNA-013 328352 461CCCGGAGACAGGGCAAGACA hypothetical miRNA-013 328353 462AAAGCGGAAACCAATCACTG mir-27b 328354 463 GTCCCCATCTCACCTTCTCT mir-27b328355 464 TCAGAGCGGAGAGACACAAG mir-96 328356 465 TAGATGCACATATCACTACCmir-17as/mir-91 328357 466 CTTGGCTTCCCGAGGCAGCT mir-17as/mir-91 328358467 AGTTTGAAGTGTCACAGCGC mir-123/mir-126as 328359 468GCGTTTTCGATGCGGTGCCG mir-123/mir-126as 328360 469 GAGACGCGGGGGCGGGGCGCmir-132 328361 470 TACCTCCAGTTCCCACAGTA mir-132 328362 471TGTGTTTTCTGACTCAGTCA mir-108-1 328363 472 AGAGCACCTGAGAGCAGCGC mir-23b328364 473 TCTTAAGTCACAAATCAGCA mir-23b 328365 474 TCTCCACAGCGGGCAATGTClet-7i 328366 475 GGCGCGCTGTCCGGGCGGGG mir-212 328367 476ACTGAGGGCGGCCCGGGCAG mir-212 328368 477 GTCCTCTTGCCCAAGCAACAhypothetical miRNA-023 328369 478 GAAGACCAATACACTCATAC mir-131-2 328370479 CCGAGGGGCAACATCACTGC let-7b 328371 480 TCCATAGCTTAGCAGGTCCA mir-1d328372 481 TTTGATAGTTTAGACACAAA mir-122a 328373 482 GGGAAGGATTGCCTAGCAGTmir-122a 328374 483 AGCTTTAGCTGGGTCAGGAC mir-22 328375 484TACCATACAGAAACACAGCA mir-92-1 328376 485 TCACAATCCCCACCAAACTC mir-92-1328377 486 TCACTCCTAAAGGTTCAAGT hypothetical miRNA-030 328378 487CACCCTCCAGTGCTGTTAGT mir-142 328379 488 CTGACTGAGACTGTTCACAG mir-183328380 489 CCTTTAGGGGTTGCCACACC hypothetical miRNA-033 328381 490ACAGGTGAGCGGATGTTCTG mir-214 328382 491 CAGACTCCCAACTGACCAGA mir-143328383 492 AGAGGGGAGACGAGAGCACT mir-192-1 328384 493TCACGTGGAGAGGAGTTAAA hypothetical miRNA-039 328385 494AGTGCTAATACTTCTTTCAT hypothetical miRNA-040 328386 495ACCTGTGTAACAGCCGTGTA hypothetical miRNA-041 328387 496TTATCGGAACTTCACAGAGA hypothetical miRNA-041 328388 497TCCCATAGCAGGGCAGAGCC let-7a-3 328389 498 GGCACTTCATTGCTGCTGCChypothetical miRNA-043 328390 499 GGAGCCTTGCGCTCAGCATT hypotheticalmiRNA-043 328391 500 ATGGTAATTTCATTTCAGGC hypothetical miRNA-044 328392501 GATTGCACATCCACACTGTC hypothetical miRNA-044 328393 502GCTGGCCTGATAGCCCTTCT mir-181a 328394 503 GTTTTTTCAAATCCCAAACT mir-181a328395 504 CCCAGTGGTGGGTGTGACCC let-7a-1 328396 505 CTGGTTGGGTATGAGACAGAmir-205 328397 506 TTGATCCATATGCAACAAGG mir-103-1 328398 507GCCATTGGGACCTGCACAGC mir-26a 328399 508 ATGGGTACCACCAGAACATG mir-33a328400 509 AGTTCAAAACTCAATCCCAA mir-196-2 328401 510GCCCTCGACGAAAACCGACT mir-196-2 328402 511 TTGAACTCCATGCCACAAGG mir-107328403 512 AGGCCTATTCCTGTAGCAAA mir-106 328404 513 GTAGATCTCAAAAAGCTACCmir-106 328405 514 CTGAACAGGGTAAAATCACT let-7f-1 328406 515AGCAAGTCTACTCCTCAGGG let-7f-1 328407 516 AATGGAGCCAAGGTGCTGCChypothetical miRNA-055 328408 517 TAGACAAAAACAGACTCTGA mir-29c 328409518 GCTAGTGACAGGTGCAGACA mir-130a 328410 519 GGGCCTATCCAAAGTGACAGhypothetical miRNA-058 328411 520 TACCTCTGCAGTATTCTACA hypotheticalmiRNA-058 328412 521 TTTACTCATACCTCGCAACC mir-218-1 328413 522AATTGTATGACATTAAATCA mir-124a-2 328414 523 CTTCAAGTGCAGCCGTAGGCmir-124a-2 328415 524 TGCCATGAGATTCAACAGTC mir-21 328416 525ACATTGCTATCATAAGAGCT mir-16-1 328417 526 TAATTTTAGAATCTTAACGC mir-16-1328418 527 AGTGTCTCATCGCAAACTTA mir-144 328419 528 TGTTGCCTAACGAACACAGAmir-221 328420 529 GCTGATTACGAAAGACAGGA mir-222 328421 530GCTTAGCTGTGTCTTACAGC mir-30d 328422 531 GAGGATGTCTGTGAATAGCC mir-30d328423 532 CCACATATACATATATACGC mir-19b-2 328424 533AGGAAGCACACATTATCACA mir-19b-2 328425 534 GACCTGCTACTCACTCTCGT mir-128b328426 535 GGTTGGCCGCAGACTCGTAC hypothetical miRNA-069 328427 536GATGTCACTGAGGAAATCAC hypothetical miRNA-070 328428 537TCAGTTGGAGGCAAAAACCC hypothetical miRNA-071 328429 538GGTAGTGCAGCGCAGCTGGT mir-29b-3 328430 539 CCGGCTATTGAGTTATGTAC mir-129-2328431 540 ACCTCTCAGGAAGACGGACT mir-133b 328432 541 GAGCATGCAACACTCTGTGChypothetical miRNA-075 328433 542 CCTCCTTGTGGGCAAAATCC let-7d 328434 543CGCATCTTGACTGTAGCATG mir-15b 328435 544 TCTAAGGGGTCACAGAAGGT mir-29a-1328436 545 GAAAATTATATTGACTCTGA mir-29a-1

Example 8 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and aDeoxy Gap Targeting pri-miRNAs

In accordance with the present invention, a second series of oligomericcompounds were designed and synthesized to target different regions ofpri-miRNA structures. The compounds are shown in Table 6. “Pri-miRNA”indicates the particular pri-miRNA which contains the miRNA that theoligomeric compound was designed to target. All compounds in Table 6 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines. The compounds can be analyzed for theireffect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-timePCR as described, supra, or they can be used in other assays toinvestigate the role of miRNAs or miRNA downstream targets.

TABLE 6 Chimeric phosphorothioate oligomeric compounds having 2′-MOEwings and a deoxy gap targeting pri-miRNAs ISIS Number SEQ ID NOSequence pri-miRNA 328637 546 GGTTCCTAATTAAACAACCC hypotheticalmiRNA-079 328638 547 CCGAGGGTCTAACCCAGCCC mir-199b 328639 548GACTACTGTTGAGAGGAACA mir-129-1 328640 549 TCTCCTTGGGTGTCCTCCTC let-7e328641 550 TGCTGACTGCTCGCCCTTGC hypothetical miRNA-083 328642 551ACTCCCAGGGTGTAACTCTA let-7c 328643 552 CATGAAGAAAGACTGTAGCC mir-204328644 553 GACAAGGTGGGAGCGAGTGG mir-145 328645 554 TGCTCAGCCAGCCCCATTCTmir-124a-1 328646 555 GCTTTTAGAACCACTGCCTC hypothetical miRNA-088 328647556 GGAGTAGATGATGGTTAGCC mir-213 328648 557 ACTGATTCAAGAGCTTTGTAhypothetical miRNA-090 328649 558 GTAGATAACTAAACACTACC mir-20 328650 559AATCCATTGAAGAGGCGATT mir-133a-1 328651 560 GGTAAGAGGATGCGCTGCTCmir-138-2 328652 561 GGCCTAATATCCCTACCCCA mir-98 328653 562GTGTTCAGAAACCCAGGCCC mir-196-1 328654 563 TCCAGGATGCAAAAGCACGAmir-125b-1 328655 564 TACAACGGCATTGTCCTGAA mir-199a-2 328656 565TTTCAGGCTCACCTCCCCAG hypothetical miRNA-099 328657 566AAAAATAATCTCTGCACAGG mir-181b 328658 567 AGAATGAGTTGACATACCAAhypothetical miRNA-101 328659 568 GCTTCACAATTAGACCATCC mir-141 328660569 AGACTCCACACCACTCATAC mir-131-1 328661 570 ATCCATTGGACAGTCGATTTmir-133a-2 328662 571 GGCGGGCGGCTCTGAGGCGG hypothetical miRNA-105 328663572 CTCTTTAGGCCAGATCCTCA hypothetical miRNA-106 328664 573TAATGGTATGTGTGGTGATA hypothetical miRNA-107 328665 574ATTACTAAGTTGTTAGCTGT mir-1b 328666 575 GATGCTAATCTACTTCACTA mir-18328667 576 TCAGCATGGTGCCCTCGCCC mir-220 328668 577 TCCGCGGGGGCGGGGAGGCThypothetical miRNA-111 328669 578 AGACCACAGCCACTCTAATC mir-7-3 328670579 TCCGTTTCCATCGTTCCACC mir-218-2 328671 580 GCCAGTGTACACAAACCAACmir-24-2 328672 581 AAGGCTTTTTGCTCAAGGGC mir-24-1 328673 582TTGACCTGAATGCTACAAGG mir-103-2 328674 583 TGCCCTGCTCAGAGCCCTAG mir-211328675 584 TCAATGTGATGGCACCACCA mir-101-3 328676 585ACCTCCCAGCCAATCCATGT mir-30b 328677 586 TCCTGGATGATATCTACCTChypothetical miRNA-120 328678 587 TCTCCCTTGATGTAATTCTA let-7a-4 328679588 AGAGCGGAGTGTTTATGTCA mir-10a 328680 589 TCATTCATTTGAAGGAAATA mir-19a328681 590 TCCAAGATGGGGTATGACCC let-7f-2 328682 591 TTTTTAAACACACATTCGCGmir-15a-1 328683 592 AGATGTGTTTCCATTCCACT mir-108-2 328684 593CCCCCTGCCGCTGGTACTCT mir-137 328685 594 CGGCCGGAGCCATAGACTCG mir-219328686 595 CTTTCAGAGAGCCACAGCCT mir-148b 328687 596 GCTTCCCAGCGGCCTATAGTmir-130b 328688 597 CAGCAGAATATCACACAGCT mir-19b-1 328689 598TACAATTTGGGAGTCCTGAA mir-199b 328690 599 GCCTCCTTCATATATTCTCA mir-204328691 600 CCCCATCTTAGCATCTAAGG mir-145 328692 601 TTGTATGGACATTTAAATCAmir-124a-1 328693 602 TTTGATTTTAATTCCAAACT mir-213 328694 603CAAACGGTAAGATTTGCAGA hypothetical miRNA-090 328695 604GGATTTAAACGGTAAACATC mir-125b-1 328696 605 CTCTAGCTCCCTCACCAGTGhypothetical miRNA-099 328697 606 GCTTGTCCACACAGTTCAAC mir-181b 328698607 GCATTGTATGTTCATATGGG mir-1b 328699 608 TGTCGTAGTACATCAGAACA mir-7-3328700 609 AGCCAGTGTGTAAAATGAGA mir-24-1 328701 610 TTCAGATATACAGCATCGGTmir-101-3 328702 611 TGACCACAAAATTCCTTACA mir-10a 328703 612ACAACTACATTCTTCTTGTA mir-19a 328704 613 TGCACCTTTTCAAAATCCAC mir-15a-1328705 614 AACGTAATCCGTATTATCCA mir-137

Example 9 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and aDeoxy Gap Targeting pri-miRNAs

In accordance with the present invention, a third series of oligomericcompounds were designed and synthesized to target different pri-miRNAstructures. The compounds are shown in Table 7. “Pri-miRNA” indicatesthe particular pri-miRNA which contains the miRNA that the oligomericcompound was designed to target. All compounds in Table 7 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.”The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds can be analyzed for their effect onmiRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR asdescribed, supra, or they can be used in other assays to investigate therole of miRNAs or miRNA downstream targets.

TABLE 7 Chimeric phosphorothioate oligomeric compounds having 2′-MOEwings and a deoxy gap targeting pri-miRNAs ISIS Number SEQ ID NOSequence pri-miRNA 328706 615 CGTGAGGGCTAGGAAATTGC mir-216 328707 616GCAACAGGCCTCAATATCTT mir-100-1 328708 617 ACGAGGGGTCAGAGCAGCGC mir-187328709 618 GGCAGACGAAAGGCTGACAG hypothetical miRNA-137 328710 619CTGCACCATGTTCGGCTCCC hypothetical miRNA-138 328711 620GGGGCCCTCAGGGCTGGGGC mir-124a-3 328712 621 CCGGTCCACTCTGTATCCAG mir-7-2328713 622 GCTGGGAAAGAGAGGGCAGA hypothetical miRNA-142 328714 623TCAGATTGCCAACATTGTGA hypothetical miRNA-143 328715 624CTGGGGAGGGGGTTAGCGTC hypothetical miRNA-144 328716 625TGGGTCTGGGGCAGCGCAGT mir-210 328717 626 TTGAAGTAGCACAGTCATAC mir-215328718 627 TCTACCACATGGAGTGTCCA mir-124a-3 328719 628AGTGCCGCTGCCGCGCCGTG mir-7-2 328720 629 ACACATTGAGAGCCTCCTGAhypothetical miRNA-142 328721 630 GTCGCTCAGTGCTCTCTAGG hypotheticalmiRNA-143 328722 631 AGGCTCCTCTGATGGAAGGT hypothetical miRNA-144 328723632 GCTGTGACTTCTGATATTAT hypothetical miRNA-153 328724 633GACATCATGTGATTTGCTCA hypothetical miRNA-154 328725 634CACCCCAAGGCTGCAGGGCA mir-26b 328726 635 TGTCAAGCCTGGTACCACCAhypothetical miRNA-156 328727 636 CTGCTCCAGAGCCCGAGTCG mir-152 328728637 ACCCTCCGCTGGCTGTCCCC mir-135-1 328729 638 TAGAGTGAATTTATCTTGGTmir-135-2 328730 639 TGGTGACTGATTCTTATCCA mir-217 328731 640CAATATGATTGGATAGAGGA hypothetical miRNA-161 328732 641TTTAAACACACATTCGCGCC mir-15a-2 328733 642 ACCGGGTGGTATCATAGACC let-7g328734 643 TGCATACCTGTTCAGTTGGA hypothetical miRNA-164 328735 644GCCCGCCTCTCTCGGCCCCC mir-33b 328736 645 TCGCCCCCTCCCAGGCCTCThypothetical miRNA-166 328737 646 ACAACTGTAGAGTATGGTCA mir-16-2 328738647 GCTGACCATCAGTACTTTCC hypothetical miRNA-168 328739 648TTATAGAACAGCCTCCAGTG hypothetical miRNA-169 328740 649TTCAGGCACTAGCAGTGGGT hypothetical miRNA-170 328741 650AGTACTGCGAGGTTAACCGC hypothetical miRNA-171 328742 651GGACCTTTAAGATGCAAAGT hypothetical miRNA-172 328743 652TTCATATTATCCACCCAGGT hypothetical miRNA-173 328744 653CGGATCCTGTTACCTCACCA mir-182 328745 654 TGGTGCCTGCCACATCTTTGhypothetical miRNA-175 328746 655 TGGGAGGCTGAATCAAGGAC hypotheticalmiRNA-176 328747 656 TGACAACCAGGAAGCTTGTG hypothetical miRNA-177 328748657 GCCAGGCAGCGAGCTTTTGA hypothetical miRNA-178 328749 658CAGCCTGCCACCGCCGCTTT hypothetical miRNA-179 328750 659CTGCCCCCGTGGACCGAACA hypothetical miRNA-180 328751 660TCGTGCACCTGAGGAGTCTG hypothetical miRNA-181 328752 661CAAACGTGCTGTCTTCCTCC mir-148a 328753 662 AAGGACTCAGCAGTGTTTCAhypothetical miRNA-183 328754 663 TCCTCGGTGGCAGAGCTCAG mir-23a 328755664 AGACAATGAGTACACAGTTC hypothetical miRNA-185 328756 665CTGCAAGCACTGGTTCCCAT hypothetical miRNA-186 328757 666TTGCCTGAGCTGCCCAAACT mir-181c 328758 667 TCCATCACACTGTCCTATGAhypothetical miRNA-188 328759 668 GAGGGATTGTATGAACATCT mir-216 328760669 GCTTGTGCGGACTAATACCA mir-100-1 328761 670 GCAGGCTAAAAGAAATAAGChypothetical miRNA-138 328762 671 ATTGTATAGACATTAAATCA mir-124a-3 328763672 GTTGAGCGCAGTAAGACAAC mir-7-2 328764 673 AGATGTTTCTGGCCTGCGAGhypothetical miRNA-142 328765 674 GACAAACTCAGCTATATTGT mir-215 328766675 ACGGCTCTGTGGCACTCATA mir-131-3 328767 676 GCTTTCTTACTTTCCACAGCmir-30c 328768 677 TACCTTTAGAATAGACAGCA mir-101-1 328769 678AGGCTGGACAGCACACAACC mir-26b 328770 679 AGCAGGAGCCTTATCTCTCChypothetical miRNA-156 328771 680 ATGAGTGAGCAGTAGAATCA mir-135-1 328772681 TGAGACTTTATTACTATCAC mir-135-2 328773 682 TACTTTACTCCAAGGTTTTAmir-15a-2 328774 683 GCACCCGCCTCACACACGTG mir-33b 328775 684TTCCCGACCTGCCTTTACCT hypothetical miRNA-166 328776 685TCCTGTAATTATAGGCTAGC hypothetical miRNA-169 328777 686GGATCATATCAATAATACCA hypothetical miRNA-172 328778 687TGCTGAGACACACAATATGT hypothetical miRNA-176 328779 688TGTTTGTCTCCAAGAAACGT hypothetical miRNA-177 328780 689TGTCATGGACAGGATGAATA hypothetical miRNA-179 328781 690TCTATCATACTCAGAGTCGG mir-148a 328782 691 TTGTGACAGGAAGCAAATCC mir-23a328783 692 CATCAGAGTCACCAACCCCA hypothetical miRNA-185 328784 693CAAGAGATGTCTCGTTTTGC hypothetical miRNA-186

Example 10 Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and aDeoxy Gap Targeted to the Stem Loop of pri-miRNA Structures

In accordance with the present invention, a fourth series of oligomericcompounds were designed to target the stem loop of different pri-miRNAstructures. In some cases, these oligomeric compounds containmismatches, and thus hybridize with partial complementarity to thestemloop structure of the pri-miRNA targeted. The compounds are shown inTable 8. “Pri-miRNA” indicates the particular pri-miRNA that theoligomeric compound was designed to target. All compounds in Table 8 arechimeric oligonucleotides (“gapmers”), composed of a central “gap”region consisting of 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by “wings.” The wings are composed of2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds can be analyzedfor their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitativereal-time PCR as described, supra, or they can be used in other assaysto investigate the role of miRNAs or downstream nucleic acid targets.

TABLE 8 Chimeric phosphorothioate compounds having 2′-MOE wings and adeoxy gap targeted to the stem loop of pri-miRNA structures Compound SEQID Number NO. Sequence Pri-miRNA RG1 694 GTGGTAGAACAGCATGACGTC mir-140RG2 695 AGCTGTGAAGCCACGATGGGC mir-30a RG3 696 AGATACAAAGATGGAAAAATCmir-29b-1 RG4 697 CTTCCTTACTATTGCTCACAA mir-34 RG5 698TGTTTAATATATATTTCACTC mir-16-3 RG6 699 TGTCAAGACATCGCGTTAACA mir-203 RG7700 TGTCGATTTAGTTATCCAACA mir-7-1 RG8 701 GTGACTATACGGATACCACAC mir-10bRG9 702 ACCTCTCCAAATGTAAAGA mir-128a RG10 703 CAAAGCGGAAACCAATCACTGmir-27b RG11 704 CTGCAGTACATGCACATATCA mir-91 RG12 705AACAATGACACCCTTGACCT mir-132 RG13 706 TTTTAATCTTAAGTCACAAA mir-23b RG14707 ATCTCCACAGCGGGCAATGTC let-7i RG15 708 TATGAAGACCAATACACTCCAmir-131-2 RG16 709 GGGGCAACATCACTGCCC let-7b RG17 710CCATGTTAGCAGGTCCATATG mir-1d RG18 711 GTTTGATAGTTTAGACACAAA mir-122aRG19 712 TGGGTCAGGACTAAAGCTTC mir-22 RG20 713 AATACCATACAGAAACACAGCmir-92-1 RG21 714 TTCGTGATGATTGTCGTGCC mir-142 RG22 715ACTGCGAGACTGTTCACAGTT mir-183 RG23 716 TACAGGTGAGCGGATGTTCTG mir-214RG24 717 TCTCAGCTCCCAACTGACCAG mir-143 RG25 718 ACCGCAGATATTACAGCCACTlet-7a-3 RG26 719 CCTGATAGCCCTTCTTAAGGA mir-181a RG27 720CTTGATCCATATGCAACAAGG mir-103-1 RG28 721 GCCATTGGGACCTGCACACC mir-26aRG29 722 GCATGGGTACCACCCCATGC mir-33a RG30 723 CGAGTTCAAAACTCAATCCCAmir-196-2 RG31 724 CTTGAACTCCATGCCACAAGG mir-107 RG32 725GTAGATCTCAAAAAGCTAGC mir-106 RG33 726 GAACAGGGTAAAATCACTAC let-7f-1 RG34727 AGACAAAAACAGACTCTGAA mir-29c RG35 728 GCTAGTGACAGGTCCAGACAG mir-130aRG36 729 TTTACTCATACCTCGCAACCA mir-218-1 RG37 730 TTAATTGTATGACATTAAATCAmir-124a-2 RG38 731 TGCCATGAGATTCAACAGTCA mir-21 RG39 732GATAATATTTAGAATCTTAAC mir-16-1 RG40 733 TAGTGTCTCATCGCAAACTTA mir-144RG41 734 CTGTTGCCTAACGAACACAGA mir-221 RG42 735 TGCTGATTACGAAAGACAGGATmir-222 RG43 736 GCTTAGCTGTGTCTTACAGCT mir-30d

Example 11 Effects of Oligomeric Compounds Targeting miRNAs on Apoptosisin Caspase Assay

Programmed cell death or apoptosis involves the activation of proteases,a family of intracellular proteases, through a cascade which leads tothe cleavage of a select set of proteins. The caspase family contains atleast 14 caspases, with differing substrate preferences. The caspaseactivity assay uses a DEVD peptide to detect activated caspases in cellculture samples. The peptide is labeled with a fluorescent molecule,7-amino-4-trifluoromethyl coumarin (AFC). Activated caspases cleave theDEVD peptide resulting in a fluorescence shift of the AFC. Increasedfluorescence is indicative of increased caspase activity andconsequently increased cell death. The chemotherapeutic drugs taxol,cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and5-fluorouracil all have been shown to induce apoptosis in acaspase-dependent manner.

The effect of several oligomeric compounds of the present invention wasexamined in cells expressing miRNA targets. The cells expressing thetargets used in these experiments were T47D, a breast carcinoma cellline. Other cell lines can also be employed in this assay and theseinclude normal human mammary epithelial cells (HMECs) as well as twobreast carcinoma cell lines, MCF7 and T47D. All of the cell lines wereobtained from the American Type Culture Collection (Manassas, Va.). Thelatter two cell lines express similar genes but MCF7 cells express thetumor suppressor p53, while T47D cells are deficient in p53. MCF-7 cellsare routinely cultured in DMEM low glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. T47D cellswere cultured in Gibco DMEM High glucose media supplemented with 10%Fetal Bovine Serum (FBS).

Cells were plated at 10,000 cells per well for HMEC cells or 20,000cells per well for MCF7 and T47D cells, and allowed to attach to wellsovernight. Plates used were 96 well Costar plate 1603 (black sides,transparent bottom). DMEM high glucose medium, with and without phenolred, were obtained from Invitrogen (San Diego, Calif.). MEGM medium,with and without phenol red, were obtained from Biowhittaker(Walkersville, Md.). The caspase-3 activity assay kit was obtained fromCalbiochem (Cat. #HTS02) (EMD Biosciences, San Diego, Calif.).

Before adding to cells, the oligomeric compound cocktail was mixedthoroughly and incubated for 0.5 hrs. The oligomeric compound or theLIPOFECTIN™-only vehicle control was added (generally from a 3 μM stockof oligonucleotide) to a final concentration of 200 nM with 6 μg/mlLIPOFECTIN™. The medium was removed from the plates and the plates weretapped on sterile gauze. Each well was washed in 150 μl of PBS (150 μLHBSS for HMEC cells). The wash buffer in each well was replaced with 100μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN™ cocktail (this wasT=0 for oligomeric compound treatment). The plates were incubated for 4hours at 37° C., after which the medium was dumped and the plate wastapped on sterile gauze. 100 μl of full growth medium without phenol redwas added to each well. After 48 hours, 50 μl of oncogene buffer(provided with Calbiochem kit) with 10 μM DTT was added to each well. 20μl of oncogene substrate (DEVD-AFC) was added to each well. The plateswere read at 400±25 nm excitation and 508±20 nm emission at t=0 and t=3time points. The t=0×(0.8) time point was subtracted from the t=3 timepoint, and the data are shown as percent of LIPOFECTIN™-only (untreatedcontrol) treated cells.

Four experiments were performed and the results are shown in Tables9-12. The concentration of oligomeric compound used was 200 nM. Allcompounds in Tables 9-12 are chimeric oligomeric compounds (“gapmers”)20 nucleotides in length, composed of a central “gap” region consistingof ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings.” The wings are composed of2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the compound. Allcytidine residues are 5-methylcytidines. As a control a 20-meroligonucleotide random-mer, ISIS-29848 (NNNNNNNNNNNNNNNNNNNN; where N isA, T, C or G; herein incorporated as SEQ ID NO: 737) was used. Inaddition, two positive controls targeting expressed genes known toinduce apoptosis when inhibited were included. These were ISIS-148715(TTGTCCCAGTCCCAGGCCTC; herein incorporated as SEQ ID NO: 738) whichtargets human Jagged2 and ISIS-226844 (GCCCTCCATGCTGGCACAGG; hereinincorporated as SEQ ID NO: 739) which targets human Notch1. Bothpositive controls have the same chemistry and gap structure as thecompounds being tested. An increase in fluorescence indicates that thecompound, by inhibiting its target, induces apoptosis as compared tountreated controls (UTC).

TABLE 9 Effects of oligomeric compounds targeting miRNAs on Apoptosis inCaspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTCUTC N/A N/A 1.0 Untreated control ISIS-29848 737 N/A 3.5 n-merISIS-148715 738 Jagged2 1.5 Positive control ISIS-226844 739 Notch1 3.6Positive control 328371 480 mir-1d 1.2 328400 509 mir-196-2 1.3 328420529 mir-222 1.0 328692 601 mir-124a-1 1.2 328381 490 mir-214 1.1 328691600 mir-145 0.9 328391 500 hypothetical miRNA-044 0.8 328415 524 mir-211.1 328433 542 let-7d 1.0 328643 552 mir-204 0.9 328377 486 hypotheticalmiRNA-030 0.7 328405 514 let-7f-1 1.0 328372 481 mir-122a 1.0 328403 512mir-106 1.0 328424 533 mir-19b-2 0.9 328648 557 hypothetical miRNA-0901.1 328397 506 mir-103-1 1.2 328656 565 hypothetical miRNA-099 1.1328392 501 hypothetical miRNA-044 1.0 328421 530 mir-30d 1.2 328417 526mir-16-1 1.0 328647 556 mir-213 0.9 328378 487 mir-142 1.0 328416 525mir-16-1 0.9

TABLE 10 Effects of oligomeric compounds targeting miRNAs on Apoptosisin Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTCUTC N/A N/A 0.9 Untreated control ISIS-29848 737 N/A 3.0 n-merISIS-148715 738 Jagged2 1.0 Positive control ISIS-226844 739 Notch1 3.1Positive control 328375 484 mir-92-1 0.9 328382 491 mir-143 0.9 328383492 mir-192-1 1.2 328385 494 hypothetical miRNA-040 0.9 328395 504let-7a-1 1.0 328398 507 mir-26a 0.9 328399 508 mir-33a 1.0 328402 511mir-107 1.2 328408 517 mir-29c 0.9 328409 518 mir-130a 0.7 328422 531mir-30d 1.0 328423 532 mir-19b-2 0.6 328425 534 mir-128b 0.8 328431 540mir-133b 0.9 328436 545 mir-29a-1 0.9 328646 555 hypothetical miRNA-0881.1 328649 558 mir-20 1.0 328651 560 mir-138-2 0.9 328652 561 mir-98 1.2328657 566 mir-181b 0.8 328672 581 mir-24-1 0.9 328694 603 hypotheticalmiRNA-090 0.8 328696 605 hypothetical miRNA-099 1.5 328700 609 mir-24-10.8

TABLE 11 Effects of oligomeric compounds targeting miRNAs on Apoptosisin Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTCUTC N/A N/A 0.9 Untreated control ISIS-29848 737 N/A 3.2 n-merISIS-148715 738 Jagged2 1.1 Positive control ISIS-226844 739 Notch1 3.1Positive control 328374 483 mir-22 1.1 328376 485 mir-92-1 0.7 328384493 hypothetical miRNA-039 1.0 328386 495 hypothetical miRNA-041 0.7328390 499 hypothetical miRNA-043 0.9 328393 502 mir-181a 1.5 328404 513mir-106 0.9 328406 515 let-7f-1 1.0 328407 516 hypothetical miRNA-0551.2 328410 519 hypothetical miRNA-058 1.5 328411 520 hypotheticalmiRNA-058 0.8 328413 522 mir-124a-2 0.8 328426 535 hypotheticalmiRNA-069 1.3 328427 536 hypothetical miRNA-070 0.8 328435 544 mir-29a-11.3 328637 546 hypothetical miRNA-079 1.0 328638 547 mir-199b 0.8 328639548 mir-129-1 0.8 328645 554 mir-124a-1 2.2 328653 562 mir-196-1 1.1328654 563 mir-125b-1 1.0 328655 564 mir-199a-2 0.7 328689 598 mir-199b0.8 328695 604 mir-125b-1 0.8

TABLE 12 Effects of oligomeric compounds targeting miRNAs on Apoptosisin Caspase Assay SEQ ID Fold Increase ISIS Number NO. Pri-miRNA over UTCUTC N/A N/A 1.0 Untreated control ISIS-29848 737 N/A 3.5 n-merISIS-148715 738 Jagged2 1.3 Positive control ISIS-226844 739 Notch1 3.5Positive control 328373 482 mir-122a 0.9 328379 488 mir-183 1.1 328387496 hypothetical miRNA-041 1.4 328388 497 let-7a-3 0.9 328389 498hypothetical miRNA-043 1.1 328394 503 mir-181a 0.8 328396 505 mir-2050.8 328401 510 mir-196-2 0.8 328412 521 mir-218-1 1.2 328414 523mir-124a-2 0.9 328418 527 mir-144 1.0 328419 528 mir-221 0.7 328430 539mir-129-2 1.3 328432 541 hypothetical miRNA-075 0.6 328434 543 mir-15b0.8 328640 549 let-7e 0.9 328641 550 hypothetical miRNA-083 1.1 328642551 let-7c 1.0 328644 553 mir-145 0.7 328650 559 mir-133a-1 0.8 328658567 hypothetical miRNA-101 1.2 328690 599 mir-204 0.8 328693 602 mir-2131.0 328697 606 mir-181b 1.0

From these data, it is evident that SEQ ID NOs. 480, 509, 601, 490, 524,557, 506, 565, 530, 605, 492, 561, 511, 555, 483, 502, 535, 562, 544,519, 516, 554, 496, 567, 521, 539, 488, 498, and 550 induce apoptosis inT47D cells, while SEQ ID NOs. 500, 486, 518, 532, 534, 566, 603, 609,485, 495, 520, 522, 536, 547, 548, 564, 598, 604, 503, 505, 510, 528,541, 543, 553, 559, and 599 prevent or have a protective effect fromapoptosis in the same system.

Example 12 Oligomeric Compounds Targeting the mir-30a pri-miRNAStructure

In one embodiment of the invention, oligomeric compounds targeting thehairpin structure of mir-30a pri-miRNA were designed and tested fortheir effects on miRNA signaling in 293T cells (American Type CultureCollection (Manassas, Va.)).

A synthetic DNA fragment comprised of four tandem repeats of the targetsite for mir-30a was cloned into the vector pGL3-C (purchased fromPromega Corp., Madison Wis.) at the unique XbaI site (pGL3C-M30-4X).This places the target site in the 3′UTR of the luciferase reportervector. An oligomeric compound mimicking the mir-30a pri-miRNA(AATTTAATACGACTCACTATAGGGCGACTGTAAACATCCTCGACTGGAAGCTGTGAAGCCACAGATGGGCTTTCAGTCGGATGTTTGCAGCTGC, herein incorporated as SEQ ID NO:1749) was in vitro transcribed using T7 RNA polymerase and a DNAtemplate produced by PCR (the T7 promoter is shown in bold).

On the day prior to the experiment 24-well plates were seeded with 293Tcells at 50% confluency. The following morning cells were treated witholigomeric compounds targeted to the mir-30a pri-miRNA mimic. Theoligomeric compounds used in this study are shown in Table 13. All ofthe compounds are 20 nucleobases in length having either aphosphorothioate backbone throughout (PS) or a phosphodiester backbonethroughout (PO). As designated in the table, ISIS 328076, 328078,328081, 328084, 328086, 328088 are chimeric oligomeric compounds(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings.” The wings arecomposed of 2′-methoxyethoxy (2′-MOE) nucleotides. All cytidine residuesare 5-methylcytidines. The remaining compounds in the table have2′-methoxyethoxy (MOE) nucleotides throughout with either aphosphorothioate (PS) or phosphodiester (PO) internucleoside linkages.

If the compound targeted the pre-loop of the mir-30a pri-miRNAstructure, that designation is also noted in the table.

TABLE 13 Oligomeric compounds targeting the mir-30a pri-miRNA Isis SEQID Number Sequence Chemistry NO 328075 GCTTCACAGCTTCCAGTCGA (PS/MOE) 740328076 GCTTCACAGCTTCCAGTCGA (PS/MOE 5-10-5 gapmer) 740 328077CCCATCTGTGGCTTCACAGC (PS/MOE); pre-loop 741 328078 CCCATCTGTGGCTTCACAGC(PS/MOE 5-10-5 gapmer); pre-loop 741 328079 CCCATCTGTGGCTTCACAGC(PO/MOE); pre-loop 741 328080 TGAAAGCCCATCTGTGGCTT (PS/MOE); pre-loop742 328081 TGAAAGCCCATCTGTGGCTT (PS/MOE 5-10-5 gapmer); pre-loop 742328082 TGAAAGCCCATCTGTGGCTT (PO/MOE); pre-loop 742 328083GCAGCTGCAAACATCCGACT (PS/MOE) 743 328084 GCAGCTGCAAACATCCGACT (PS/MOE5-10-5 gapmer) 743 328085 CATCTGTGGCTTCACAGCTT (PS/MOE) 744 328086CATCTGTGGCTTCACAGCTT (PS/MOE 5-10-5 gapmer) 744 328087AAGCCCATCTGTGGCTTCAC (PS/MOE) 745 328088 AAGCCCATCTGTGGCTTCAC (PS/MOE5-10-5 gapmer) 745

Cells were washed once with PBS then oligomeric compounds were added totriplicate wells at 150 nM in OPTI-MEM™ media and 4.5 μl/ml LIPOFECTIN™reagent (Invitrogen Corporation, Carlsbad, Calif.). After 3 hours, themedia was removed, and the cells were treated with the mir-30a pri-miRNAmimic at 200 nM in OPTI-MEM™ with 6 μl/ml LIPOFECTIN™ reagent. After 3hours the media was removed from the cells. The reporter plasmid,pGL3C-M30-4X, was then transfected using SuperFect reagent. 20 μg ofpGL3C-M30-4X and 2 μg of pRL-CMV, a plasmid expressing Renillaluciferase, were suspended in 600 μL of serum-free DMEM to which 120 μlof Superfect was added. After a 5 minute incubation, 6 mls of DMEM+10%FCS was added. 125 μL of the plasmid/SuperFect suspension was added toeach well. After a 2 hour incubation cells were washed and fresh growthmedia added. Cells were incubated overnight.

The following morning the media was removed and the cells were lysed in120 μL passive lysis buffer (PLB; Promega). 40 μL of the lysate was thenassayed for Photinus (PL) and Renilla (RL) luciferases using a DualLuciferase Assay kit (Promega) according to the manufacturer's protocol.The results below are given as percent pGL3C-M30-4× expression (PL)normalized to pRL-CMV expression (RL). The 20-nucleobase oligonucleotiderandom-mer ISIS Number 29848 was used as a negative control. The dataare shown in Table 14.

TABLE 14 Effects of oligomeric compounds targeting the mir-30a pri-miRNAon reporter gene expression percent control SEQ ID luciferase NO ISISNumber expression N/A Untreated control 100 N/A Mir-30a pri-miRNA only62 737 29848 control added after mir-30a pri-miRNA 63 292 327874 66 740328075 55 740 328076 57 741 328077 70 741 328078 63 742 328080 72 742328081 80 743 328084 75 744 328085 72 744 328086 95 745 328087 83 745328088 107

Upon administration of the mir-30a pri-miRNA mimic, the pri-miRNA isbelieved to be processed in the cell by the endogenous Drosha RNase IIIenzyme into a pre-miRNA, which is then processed by human Dicer into amature miRNA, which is then able to hybridize to the target site, thuseffectively reducing luciferase reporter expression.

Upon treatment of the system with the oligomeric compounds targeting themir-30a pri-miRNA, the processing and/or production of the mir-30amature miRNA is inhibited, and the mir-30a miRNA is no longer able tobind its target site, thus allowing luciferase reporter expression toincrease.

Cells treated with mir-30a pri-miRNA mimic show an approximately 38%reduction in luciferase expression compared to the untreated controls.Treatment with ISIS 328086, 328087 and 328088 had the most dramaticeffect in reversing the mir-30a miRNA-mediated silencing, restoringluciferase reporter expression to near control levels. Thus, it wasdemonstrated that the oligomeric compound mimicking the mir-30apri-miRNA silences luciferase activity from the reporter vector, andthat oligomeric compounds targeting the mir-30a pri-miRNA can inhibitits silencing activity, possibly by interfering with its processing intothe pre-miRNA or mature miRNA molecules.

ISIS 328085 to ISIS 328088 were designed to target the mir-30a pri-miRNAas pseudo half-knot compounds. Methods for the preparation of pseudohalf-knot compounds are disclosed in U.S. Pat. No. 5,512,438 which isincorporated herein by reference. This motif has been used to disruptthe structure of regulatory RNA stem loops in larger viral genomicstructures. (Ecker et al, Science. 1992; 257:958-61). However, this isthe first example of the pseudo half-knot motif being used to regulate asmall non-coding RNA, more specifically a miRNA such as those disclosedherein. It is also the first demonstration of apoptotic modulation in acell by pseudo half-knot structured oligomeric compounds.

Example 13 Effects of Oligomeric Compounds Targeting miRNAs onExpression of Adipocyte Differentiation Markers

The effect of several oligomeric compounds of the present inventiontargeting miRNAs on the expression of markers of cellulardifferentiation was examined in preadipocytes.

One of the hallmarks of cellular differentiation is the upregulation ofgene expression. During adipocyte differentiation, the gene expressionpatterns in adipocytes change considerably. An excessive recruitment anddifferentiation of preadipocytes into mature adipocytes is acharacteristic of human obesity, which is a strong risk factor for type2 diabetes, hypertension, atherosclerosis, cardiovascular disease, andcertain cancers. Some genes known to be upregulated during adipocytedifferentiation include hormone-sensitive lipase (HSL), adipocyte lipidbinding protein (aP2), glucose transporter 4 (Glut4), and PPARγ(Peroxisome proliferator-activated receptor gamma). These genes playimportant roles in the uptake of glucose and the metabolism andutilization of fats. For example, HSL is involved in the mobilization offatty acids from adipose tissue into the bloodstream; studies suggestthat increased free fatty acid levels are one of the causative factorsin type 2 diabetes. aP2 is believed to play a role in athersclerosis.Glut4 is important in insulin signaling. PPARγ is believed to beinvolved in adipocyte differentiation, insulin sensitivity, and colonictumor development.

Leptin is also a marker for differentiated adipocytes. In the adipocyteassay, leptin secretion into the media above the differentiatedadipocytes was measured by protein ELISA. Cell growth, transfection anddifferentiation procedures were carried out as described for theTriglyceride accumulation assay (see below). On day ninepost-transfection, 96-well plates were coated with a monoclonal antibodyto human leptin (R&D Systems, Minneapolis, Minn.) and left at 4° C.overnight. The plates were blocked with bovine serum albumin (BSA), anda dilution of the media was incubated in the plate at RT for 2 hours.After washing to remove unbound components, a second monoclonal antibodyto human leptin (conjugated with biotin) was added. The plate was thenincubated with strepavidin-conjugated horseradish peroxidase (HRP) andenzyme levels are determined by incubation with 3, 3′, 5,5′-Tetramethlybenzidine, which turns blue when cleaved by HRP. The OD₄₅₀was read for each well, where the dye absorbance is proportional to theleptin concentration in the cell lysate. Results are expressed as apercent±standard deviation relative to transfectant-only controls.

An increase in triglyceride content is another well-established markerfor adipocyte differentiation. The triglyceride accumulation assaymeasures the synthesis of triglyceride by adipocytes. Triglycerideaccumulation was measured using the Infinity™ Triglyceride reagent kit(Sigma-Aldrich, St. Louis, Mo.). Human white preadipocytes (Zen-BioInc., Research Triangle Park, N.C.) were grown in preadipocyte media(ZenBio Inc.). One day before transfection, 96-well plates were seededwith 3000 cells/well. Cells were transfected according to standardpublished procedures with 250 nM oligomeric compound in LIPOFECTIN™(Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem.1993 268(19):14514-22). Oligomeric compounds were tested in triplicateon each 96-well plate, and the effects of TNF-α, a positive drug controlthat inhibits adipocyte differentiation, were also measured intriplicate. Negative and transfectant-only controls may be measured upto six times per plate. After the cells have reached confluence(approximately three days), they were exposed to differentiation media(Zen-Bio, Inc.) containing a PPAR-γ agonist, IBMX, dexamethasone, andinsulin for three days. Cells were then fed adipocyte media (Zen-Bio,Inc.), which was replaced at 2 to 3 day intervals. On day ninepost-transfection, cells were washed and lysed at room temperature, andthe triglyceride assay reagent was added. Triglyceride accumulation wasmeasured based on the amount of glycerol liberated from triglycerides bythe enzyme lipoprotein lipase. Liberated glycerol is phosphorylated byglycerol kinase, and hydrogen peroxide is generated during the oxidationof glycerol-1-phosphate to dihydroxyacetone phosphate by glycerolphosphate oxidase. Horseradish peroxidase (HRP) uses H₂O₂ to oxidize4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to producea red-colored dye. Dye absorbance, which is proportional to theconcentration of glycerol, was measured at 515 nm using an UVspectrophotometer. Glycerol concentration was calculated from a standardcurve for each assay, and data were normalized to total cellular proteinas determined by a Bradford assay (Bio-Rad Laboratories, Hercules,Calif.). Results are expressed as a percent±standard deviation relativeto transfectant-only control.

For assaying adipocyte differentiation, expression of the four hallmarkgenes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG)accumulation and leptin secretion were measured in adipocytestransfected with the uniform 2′-MOE phosphorothioate (PS) oligomericcompounds previously described. Cells are lysed on day ninepost-transfection, in a guanadinium-containing buffer and total RNA isharvested. Real-time PCR is performed (Applied Biosystems, Prism 7700)on the total RNA using the following primer/probe sets for the adipocytedifferentiation hallmark genes: (aP2): forward 5′-GGTGGTGGAATGCGTCATG-3′(SEQ ID NO: 746), reverse 5′-CAACGTCCCTTGGCTTATGC-3′ (SEQ ID NO: 747),probe 5′-FAM-AAGGCGTCACTTCCACGAGAGTTTATGAGA-TAMRA-3′ (SEQ ID NO: 748);(Glut4): forward 5′-GGCCTCCGCAGGTTCTG-3′ (SEQ ID NO: 749), reverse5′-TTCGGAGCCTATCTGTTGGAA-3′ (SEQ ID NO: 750), probe5′-FAM-TCCAGGCCGGAGTCAGAGACTCCA-TAMRA-3′ (SEQ ID NO: 751); (HSL):forward 5′-ACCTGCGCACAATGACACA-3′ (SEQ ID NO: 752), reverse5′-TGGCTCGAGAAGAAGGCTATG-3′ (SEQ ID NO: 753), probe5′-FAM-CCTCCGCCAGAGTCACCAGCG-TAMRA-3′ (SEQ ID NO: 754); (PPAR-γ):forward 5′-AAATATCAGTGTGAATTACAGCAAACC-3′ (SEQ ID NO: 755), reverse5′-GGAATCGCTTTCTGGGTCAA-3′ (SEQ ID NO: 756), probe5′-FAM-TGCTGTTATGGGTGAAACTCTGGGAGATTCT-TAMRA-3′ (SEQ ID NO: 757). Theamount of total RNA in each sample is determined using a Ribogreen Assay(Molecular Probes, Eugene, Oreg.), and expression levels of theadipocyte differentiation hallmark genes were normalized to total RNA.Leptin protein and triglyceride levels as well as mRNA levels for eachof the four adipocyte differentiation hallmark genes are expressedrelative to control levels (control=treatment with ISIS-29848 (SEQ IDNO: 737)). Results of two experiments are shown in Tables 15 and 16.

TABLE 15 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers ISIS SEQ ID Number NO TG AP2 HSLGlut4 PPAR gamma 327876 294 0.47 0.75 0.47 0.36 0.57 327878 296 0.650.85 0.93 0.69 0.97 327880 298 0.52 0.97 0.80 1.11 0.53 327888 306 0.981.18 1.38 1.37 1.36 327889 307 0.47 0.69 0.59 0.55 0.71 327890 308 0.920.91 0.86 1.10 1.18 327892 310 0.42 0.31 0.25 0.07 0.32 327901 319 0.540.42 0.33 0.19 0.30 327903 321 1.20 1.15 1.23 1.72 1.19 327905 323 0.691.14 1.11 0.84 0.54 327913 331 0.59 0.99 0.92 0.84 0.72 327919 337 0.580.79 0.57 0.32 0.52 327922 340 1.09 0.99 0.95 1.75 1.37 327925 343 0.720.77 0.78 1.99 0.60 327933 351 1.48 1.46 1.35 2.52 1.52 327934 352 0.991.20 1.02 1.22 0.97 327939 357 0.92 1.08 1.21 0.87 0.83 327941 359 1.311.78 1.73 2.07 0.80 327954 372 0.58 0.95 1.03 0.92 0.73

TABLE 16 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers ISIS SEQ ID Number NO TG Leptin AP2HSL Glut4 PPAR gamma 327888 306 0.44 1.38 0.47 0.50 0.17 0.66 327889 3070.46 1.05 0.57 0.54 0.46 0.82 327890 308 0.61 1.36 0.69 0.67 0.67 0.94327893 311 0.95 1.14 0.97 0.85 1.47 1.03 327901 319 0.53 1.02 0.47 0.470.29 0.72 327903 321 0.58 1.61 0.92 0.80 1.12 0.98 327905 323 0.58 1.620.68 0.69 0.40 0.83 327919 337 0.40 1.44 0.48 0.37 0.18 0.57 327922 3400.43 1.25 0.75 0.72 0.43 0.80 327925 343 0.63 1.40 0.77 0.75 0.61 0.83327926 344 1.06 1.47 0.85 0.82 1.10 0.93 327930 348 0.97 0.95 0.86 0.891.01 0.98 327931 349 1.11 1.12 1.00 0.99 1.37 1.56 327934 352 0.62 1.250.66 0.64 0.44 0.72 327938 356 1.05 1.35 0.86 0.85 0.80 0.90 327939 3570.59 2.67 0.69 0.63 0.30 0.70 327941 359 0.42 0.54 0.88 0.81 0.44 0.86327942 360 0.85 2.03 0.82 0.79 0.66 0.87 327955 373 0.81 1.22 0.74 0.820.45 0.92 327967 385 0.90 1.22 0.86 0.97 0.56 0.89

From these data, values above 1.0 for triglyceride accumulation (column“TG” in the tables) indicate that the compound has the ability tostimulate triglyceride accumulation, whereas values at or below 1.0indicate that the compound inhibits triglyceride accumulation. Withrespect to leptin secretion (column “Leptin” in the tables), valuesabove 1.0 indicate that the compound has the ability to stimulatesecretion of the leptin hormone, and values at or below 1.0 indicatethat the compound has the ability to inhibit secretion of leptin. Withrespect to the four adipocyte differentiation hallmark genes (columns“AP2,” “HSL,” “Glut4,” and “PPAR gamma” in the tables), values above 1.0indicate induction of cell differentiation, whereas values at or below1.0 indicate that the compound inhibits differentiation.

Several compounds were found to have remarkable effects. For example,the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targetedto mir-23b; ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131-1,mir-131-2 and mir-131-3 (also known as mir-9); ISIS Number 327942 (SEQID NO: 360) targeted to mir-141 and ISIS Number 327901 (SEQ ID NO: 319),targeted to mir-143 were shown to significantly reduce the expressionlevels of 5 of 6 markers of adipocyte differentiation (excepting leptinlevels), indicating that these oligomeric compounds have the ability toblock adipocyte differentiation. Therefore, these oligomeric compoundsmay be useful as pharmaceutical agents with applications in thetreatment, attenuation or prevention of obesity, hyperlipidemia,atherosclerosis, atherogenesis, diabetes, hypertension, or othermetabolic diseases as well as having potential applications in themaintenance of the pluripotent phenotype of stem or precursor cells.

The compound ISIS Number 327939 (SEQ ID NO: 357), targeted tomir-125b-1, for example, produced surprising results in that itdemonstrates a significant increase in leptin secretion but aconcomitant decrease in triglyeride accumulation and a decrease in theexpression of all four adipocyte differentiation hallmark genes,indicating that this oligomeric compound may be useful as a pharmaceuticagent in the treatment of obesity, as well as having applications inother metabolic diseases.

The oligomeric compound ISIS Number 327931 (SEQ ID NO: 349), targeted tolet-7c is an example of a compound which demonstrates an increase infour out of six markers of adipocyte differentiation, including asignificant increase in the expression of PPAR-γ. This oligomericcompound may be useful as a pharmaceutical agent in the treatment ofdiseases in which the induction of cell differentiation is desirable.

The oligomeric compound ISIS Number 327933 (SEQ ID NO: 351), targeted tomir-145 is an example of a compound which demonstrates an increase inall six markers of adipocyte differentiation. This oligomeric compoundmay be useful as a pharmaceutical agent in the treatment of diseases inwhich the induction of adipocyte differentiation is desirable, such asanorexia, or for conditions or injuries in which the induction ofcellular differentiation is desirable, such as Alzheimers disease orcentral nervous system injury, in which regeneration of neural tissue(such as from pluripotent stem cells) would be beneficial. Furthermore,this oligomeric compound may be useful in the treatment, attenuation orprevention of diseases in which it is desirable to induce cellulardifferentiation and/or quiescence, for example in the treatment ofhyperproliferative disorders such as cancer.

In some embodiments, differentiating adipocytes were treated withuniform 2′-MOE phosphorothioate oligomeric compounds according to themethods described above, and the expression of the four hallmark genes,HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulationwere measured. TG levels as well as mRNA levels for each of the fouradipocyte differentiation hallmark genes are expressed as a percentageof control levels (control=treatment with ISIS 342673;AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), auniform 2′-MOE phosphorothioate oligomeric compound containing 15mismatches with respect to the mature mir-143 miRNA). Undifferentiatedadipocytes were also compared as a negative control. As a positivecontrol, differentiating adipocytes were treated with ISIS 105990(AGCAAAAGATCAATCCGTTA; herein incorporated as SEQ ID NO: 759), a 5-10-5gapmer oligomeric compound targeting the PPAR-gamma mRNA, previouslydemonstrated to inhibit adipocyte differentiation. The effects of TNF-α,also known to inhibit adipocyte differentiation, were also measured.Results of these experiments are shown in Tables 17 and 18.

TABLE 17 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers ISIS SEQ Number ID NO TG AP2 HSLGlut4 PPAR gamma Untreated N/A 88.5 87.8 88.6 102.7 94.9 control 105990759 28.2 51.6 49.2 59.5 51.8 342673 758 100.0 100.0 100.0 100.0 100.0TNF-alpha N/A 10.0 5.5 0.7 0.5 18.8 Undiff. N/A 2.7 0.0 0.3 0.1 9.2adipocytes 328116 418 82.1 87.7 75.8 75.2 78.4 328117 419 55.0 65.4 61.768.1 64.1 328118 420 69.3 92.7 85.3 76.6 80.2 328119 421 90.2 99.9 98.595.2 82.7 328120 422 82.7 81.0 77.7 94.8 70.5 328121 423 134.8 127.0126.0 140.8 103.6 328122 424 78.9 79.3 72.7 85.9 77.8 328123 425 120.8106.7 85.4 162.4 74.7 328124 426 99.1 101.8 103.6 122.7 90.4 328125 42781.7 86.9 75.8 99.5 76.1 328126 428 98.9 90.9 83.2 100.7 75.0 328127 42974.5 86.9 89.7 80.8 77.6 328128 430 98.7 100.7 94.1 101.9 84.0 328129431 53.8 67.6 56.5 60.0 71.8 328130 432 122.4 86.6 76.5 83.8 99.4 328131433 89.1 95.4 81.8 103.6 88.2 328132 434 114.1 90.2 73.7 72.1 90.0328133 435 61.2 69.5 63.0 91.9 63.8 328134 436 85.7 80.1 74.7 88.3 78.4328135 437 63.6 80.6 76.7 90.3 70.0 328136 438 47.0 73.0 65.0 66.7 72.7328137 439 83.2 99.6 86.3 88.5 85.7 328138 440 100.6 85.3 89.8 86.8 83.8328139 441 89.1 98.3 92.6 106.3 115.0

TABLE 18 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers SEQ ISIS # ID NO TG AP2 HSL Glut4PPAR gamma Untreated N/A 102.2 90.8 94.9 117.8 103.3 control 105990 75932.8 49.8 52.0 68.1 60.1 342673 758 100 100 100 100 100 TNF-alpha N/A14.5 9.6 3.1 1.9 27.9 Undiff. N/A 2.8 0.0 1.4 0.3 10.7 adipocytes 327912330 107.4 90.1 90.6 89.0 76.9 327969 387 46.0 59.8 66.4 60.6 69.2 328099401 93.9 85.9 88.4 86.8 81.9 328100 402 71.5 61.9 72.0 74.2 66.7 328101403 108.6 83.2 91.8 84.7 79.3 328102 404 95.9 87.9 97.0 79.2 93.7 328103405 110.2 83.2 82.5 94.3 74.3 328104 406 122.6 102.2 98.2 119.1 90.4328105 407 93.1 88.2 94.2 94.2 93.3 328106 408 90.5 88.8 94.9 105.7 90.7328107 409 66.7 67.5 61.0 72.5 79.3 328108 410 89.6 83.7 90.1 94.9 84.0328109 411 84.9 84.9 86.9 106.6 96.1 328110 412 97.7 93.3 91.0 104.791.2 328111 413 101.9 71.5 69.5 59.6 74.9 328112 414 98.1 99.1 101.2122.5 102.4 328113 415 80.8 84.5 90.6 99.9 93.8 328114 416 117.3 94.493.3 114.9 89.3 328115 417 108.7 80.0 89.0 132.0 95.8 341803 760 85.977.3 75.5 86.8 71.2 341804 761 60.9 70.8 71.6 73.6 74.1 341805 762 78.181.9 81.8 88.2 80.4 341806 763 83.2 75.8 73.4 69.4 72.6 341807 764 114.174.8 96.8 119.5 86.2

Several compounds were found to have remarkable effects. For example,the oligomeric compounds ISIS Number 328117 (SEQ ID NO: 419), targetedto hypothetical miRNA-144, ISIS Number 328129 (SEQ ID NO: 431), targetedto hypothetical miRNA-173, ISIS Number 328136 (SEQ ID NO: 438), targetedto hypothetical miRNA-181, and ISIS Number 327969 (SEQ ID NO: 387),targeted to mir-182, were each shown to reduce the expression levels oftriglycerides by at least 50%, and treatment with ISIS 328117, 328129,or 328136 also each resulted in a reduction of expression of the otherfour hallmark genes, indicating that these oligomeric compounds targetedto hypothetical miRNA-144, hypothetical miRNA-173, hypotheticalmiRNA-181, and mir-182, may be useful as therapeutic agents withapplications in the treatment, attenuation or prevention of obesity,hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension,or other metabolic diseases.

The oligomeric compound ISIS Number 328121 (SEQ ID NO: 423), targeted tohypothetical miRNA-161 is an example of a compound which stimulates anincrease in all five markers of adipocyte differentiation. Thisoligomeric compound may be useful as a pharmaceutical agent in thetreatment of diseases in which the induction of adipocytedifferentiation is desirable, such as anorexia, or for conditions orinjuries in which the induction of cellular differentiation isdesirable, such as Alzheimers disease or central nervous system injury,in which regeneration of neural tissue would be beneficial. Furthermore,this oligomeric compound may be useful in the treatment, attenuation orprevention of diseases in which it is desirable to induce cellulardifferentiation and/or quiescence, for example in the treatment ofhyperproliferative disorders such as cancer.

Example 14 Expression of mir-143 in Human Tissues and Cell Lines

Total RNA from spleen, kidney, testicle, heart and liver tissues as wellas total RNA from human promyelocytic leukemia HL-60 cells, humanembryonic kidney 293 (HEK293) cells, and T47D human breast carcinomacells was purchased from Ambion, Inc. (Austin, Tex.). RNA frompreadipocytes and differentiated adipocytes was purchased from Zen-Bio,Inc. (Research Triangle Park, N.C.). RNA was prepared from the HeLa,NT2, T-24, and A549 cell lines cultured as described above, using thefollowing protocol: cell monolayers were washed twice with cold PBS, andcells were lysed in 1 mL TRIZOL™ (Invitrogen) and total RNA preparedusing the manufacturer's recommended protocols.

Fifteen to twenty micrograms of total RNA was fractionated byelectrophoresis through 10% acrylamide urea gels using a TBE buffersystem (Invitrogen). RNA was transferred from the gel to HYBOND™-N+nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) byelectroblotting in an Xcell SureLock™ Minicell (Invitrogen). Membraneswere fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400(Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hybbuffer solution (Amersham) using manufacturer's recommendations foroligonucleotide probes.

To detect mir-143, a target specific DNA oligonucleotide probe with thesequence TGAGCTACAGTGCTTCATCTCA (SEQ ID NO: 319) was synthesized by IDT(Coralville, Iowa). The oligo probe was 5′ end-labeled with T4polynucleotide kinase with (γ-³²P) ATP (Promega). To normalize forvariations in loading and transfer efficiency membranes can be strippedand probed for U6 RNA. Hybridized membranes were visualized andquantitated using a Storm 860 PHOSPHORIMAGER™ and IMAGEQUANT™ SoftwareV3.3 (Molecular Dynamics, Sunnyvale, Calif.).

Using this probe, the mir-143 miRNA was found to be most highlyexpressed in human heart, thymus and kidney, and was also expressed to alesser extent in lung, spleen, liver, and brain tissues. For example, ascompared to expression levels in liver, mir-143 was expressedapproximately 24-fold higher in heart, 17-fold higher in thymus, and8-fold higher in kidney.

The mir-143 miRNA was also found to be expressed in adipocytes andpreadipocytes, and levels of mir-143 were found to be dramaticallyupregulated in differentiated adipocytes as compared to preadipocytes,indicating that this miRNA may be important in adipocytedifferentiation. These data, taken together with the finding that theoligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), targeted tomir-143, was shown to inhibit the adipocyte differentiation markers(described above, Example 13), supports the conclusion that mir-143 isinvolved in cellular differentiation pathways.

Example 15 Effects of Oligomeric Compounds Targeting miRNAs on Apoptosisin the Caspase Assay in Preadipocytes

The effect of oligomeric compounds of the present invention targetingmiRNAs was examined in preadipocytes (Zen-Bio, Inc., Research TrianglePark, N.C.) using the fluorometric caspase assay previously described inExample 11. The oligonucleotide random-mer, ISIS-29848 (SEQ ID NO: 737)was used as a negative control, and ISIS-148715 (SEQ ID NO: 738),targeting the human Jagged2 mRNA, known to induce apoptosis wheninhibited, was used as a positive control. The measurement obtained fromthe untreated control cells is designated as 100% activity and was setequal to 1.0. Results are shown in Table 19.

TABLE 19 Effects of targeting miRNAs on apoptosis in preadipocytes SEQID ISIS Number NO. Pri-miRNA Fold Increase over UTC UTC N/A N/A 1.0Untreated control ISIS-29848 737 N/A 1.2 n-mer ISIS-148715 738 Jagged236.9 Positive control 327888 306 mir-108-1 1.1 327889 307 mir-23b 1.1327890 308 let-7i 1.3 327893 311 let-7b 1.3 327901 319 mir-143 2.0327903 321 let-7a-3 1.6 327905 323 mir-205 1.5 327919 337 mir-221 1.3327922 340 mir-19b-2 1.0 327925 343 mir-133b 2.0 327926 344 let-7d 1.8327930 348 let-7e 1.4 327931 349 let-7c 1.5 327934 352 mir-213 2.0327938 356 mir-98 1.0 327939 357 mir-125b-1 2.2 327941 359 mir-181b 1.3327942 360 mir-141 1.0 327955 373 mir-130b 4.3 327967 385 let-7g 1.5

From these data, it is evident that the oligomeric compounds of thepresent invention generally do not induce the activity of caspasesinvolved in apoptotic pathways in preadipocytes. In particular, theoligomeric compound targeting mir-143, ISIS Number 327901 (SEQ ID NO:319), does not result in a significant increase in caspase activity ascompared to the Jagged2 positive control. Taken together with theresults from the adipocyte differentiation assay (Example 13) and theexpression analysis of mir-143 (Example 14), these data suggest that themir-143 miRNA plays a role in stimulating cellular differentiation,employing pathways other than the caspase cascades activated duringapoptosis.

It was recently reported that bone marrow cells may contribute to thepathogenesis of vascular diseases, and that cell differentiation appearsto be important in models of postangioplasty restenosis, graftvasculopathy, and hyperlipidemia-induced atherosclerosis. Bone marrowcells have the potential to give rise to vascular progenitor cells thathome in on damaged vessels and differentiate into smooth muscle cells orendothelial cells, thereby contributing to vascular repair, remodeling,and lesion formation (Sata, M. Trends Cardiovasc Med. 200313(6):249-53). Thus, the ability to modulate cell differentiation mayprovide the basis for the development of new therapeutic strategies forvascular diseases, targeting mobilization, homing, differentiation, andproliferation of circulating vascular progenitor cells.

Example 16 Comparison of Effects of Oligomeric Compounds Targeting themir-143 pri-miRNA or Mature mir-143 miRNA on Adipocyte Differentiation

Two oligomeric compounds targeting the mature mir-143 miRNA and twooligomeric compounds targeting the 110-nucleotide mir-143 pri-miRNA werecompared for their effects on adipocyte differentiation using the sameadipocyte differentiation assay as described in Example 13.

The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319),22-nucleotides in length, targets the mature mir-143 miRNA and iscomposed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate(P═S) internucleoside (backbone) linkages throughout. The oligomericcompound ISIS Number 338664 (CAGACTCCCAACTGACCAGA; SEQ ID NO: 491) isalso a uniform 2′-MOE oligonucleotide, which is designed to target themir-143 pri-miRNA. Another oligomeric compound targeting the mir-143pri-miRNA, ISIS Number 328382 (SEQ ID NO: 491) is a chimericoligonucleotide, 20 nucleotides in length, composed of a central “gap”region consisting of ten 2′-deoxynucleotides, which is flanked on bothsides (5′ and 3′ directions) by five-nucleotide “wings” having 2′-MOEsubstituents in the wing nucleosides (a “5-10-5 gapmer”), and ISISNumber 340927 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319) is a 5-10-7gapmer designed to target mature mir-143. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The effect of these oligomericcompounds targeting the mir-143 miRNA and the mir-143 pri-miRNA onexpression of the 5 hallmark genes indicating cellular differentiationwas examined in preadipocytes using the same methods described inExample 13. Results are shown in Table 20.

TABLE 20 Comparison of uniform 2′-MOE and chimeric oligomeric compoundstargeting the mir-143 miRNA and pri-miRNAs on expression of adipocytedifferentiation markers ISIS SEQ ID Number NO TG AP2 HSL Glut4 PPARgamma 327901 319 0.54 0.42 0.33 0.19 0.30 328382 491 0.72 0.89 0.75 0.850.96 338664 491 1.42 1.01 0.76 1.81 0.86 340927 319 0.65 0.77 0.73 0.540.36

From these data, it was observed that while the gapmer oligomericcompound targeting the mature mir-143 (ISIS Number 340972) results inreduced expression of the adipocyte differentiation markers, the uniform2′-MOE oligomeric compound targeting mature mir-143 (ISIS Number 327901)was more effective. For the oligomeric compounds targeting the mir-143pri-miRNA, the gapmer compound (ISIS Number 328382) appeared to be moreeffective in blocking adipocyte differentiation than was the uniform2′-MOE oligomeric compound (ISIS Number 338664).

Dose Responsiveness:

In one embodiment, the oligomeric compound ISIS Number 327901 (SEQ IDNO: 319) targeting mature mir-143 was selected for additional doseresponse studies in the adipocyte differentiation assay. Differentiatingadipocytes (at day 10 post-induction of differentiation) were treatedwith 50, 100, 200, and 300 nM ISIS 327901, or the scrambled control ISISNumber 342673 (SEQ ID NO: 758) containing 15 mismatches with respect tothe mature mir-143 miRNA. ISIS Numbers 327901 and 342673 are uniform2′-MOE phosphorothioate oligomeric compounds 22 nucleotides in length.Differentiating adipocytes treated with ISIS Number 29848 (SEQ ID NO:737) served as the negative control to which the data were normalized.Differentiating adipocytes treated with ISIS 105990 (SEQ ID NO: 759), a5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA whichhas been demonstrated previously to inhibit adipocyte differentiation,served as the positive control. Triglyceride levels as well as mRNAlevels for each of the four adipocyte differentiation hallmark genes(PPAR-gamma, aP2, HSL, and GLUT4) were measured 24 hours after treatmentas described above. Untreated cells were compared to cells treated witholigomeric compounds, and results of these dose response studies areshown in Table 21, where levels of the markers is expressed as apercentage of untreated control (% UTC) levels. Where present, “N.D.”indicates “no data.”

TABLE 21 Effects of oligomeric compounds targeting mir-143 on expressionof adipocyte differentiation markers % UTC Hallmark Dose of oligomericcompound Measured: Isis #: 50 nM 100 nM 200 nM 300 nM Triglycerides342673 94.2 105.3  98.3 108.2 negative control 105990 N.D. N.D. N.D.16.6 positive control 327901 85.3 68.9 34.0 23.0 PPAR-gamma 342673 77.589.9 94.6 85.8 mRNA negative control 105990 N.D. N.D. N.D. 43.9 positivecontrol 327901 74.6 70.8 51.8 39.3 AP2 mRNA 342673 82.4 90.3 81.1 70.9negative control 105990 N.D. N.D. N.D. 17.9 positive control 327901 78.364.6 39.0 22.4 HSL mRNA 342673 92.0 95.6 97.3 85.2 negative control105990 N.D. N.D. N.D. 7.4 positive control 327901 89.5 73.5 40.2 11.9GLUT4 mRNA 342673 94.9 90.7 97.6 102.7 negative control 105990 N.D. N.D.N.D. 11.8 positive control 327901 74.2 49.7 32.8 17.4

From these data, it was observed that treatment of differentiatingadipocytes with the uniform 2′-MOE oligomeric compound, ISIS Number327901 targeting mir-143 results in a dose responsive reduction ofexpression of all five markers of differentiation. Thus, this oligomericcompound may be useful in the treatment of diseases associated withincreased expression of these hallmark genes, such as obesity,hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension,or other metabolic diseases as well as having potential applications inthe maintenance of the pluripotent phenotype of stem or precursor cells.

Example 17 Human Let 7 Homologs

Let-7 is one of the two miRNAs originally identified in C. elegans as anantisense translational repressor of messenger RNAs encoding keydevelopmental timing regulators in nematode larva. Several genespredicted to encode let-7-like miRNAs have been identified in a widevariety of species, and these let-7-like homologs are believed tocontrol temporal transitions during development across animal phylogeny.Oligomeric compounds of the present invention were designed to targetseveral human let-7-like genes. Additionally, a series oftarget-specific DNA oligonucleotide probes were synthesized by IDT(Coralville, Iowa) and used in Northern analyses to assess theexpression of let-7-like miRNA homologs in various tissues. These let-7homolog specific probes are shown in Table 22.

TABLE 22 Probes for Northern analyses of mRNA expression of let-7homologs SEQ ID ISIS Number NO Sequence pri-miRNA 327890 308AGCACAAACTACTACCTCA let-7i 327893 311 AACCACACAACCTACTACCTCA let-7b327903 321 AACTATACAACCTACTACCTCA let-7a-3 327926 344ACTATGCAACCTACTACCTCT let-7d 327930 348 ACTATACAACCTCCTACCTCA let-7e327931 349 AACCATACAACCTACTACCTCA let-7c 327967 385ACTGTACAAACTACTACCTCA let-7g

For Northern analyses with let-7 homolog probes, total RNA from spleen,kidney, testes, heart, and liver tissues as well as total RNA fromHEK293, T47D, T-24, MCF7, HepG2, and K-562 Leukemia cell lines waseither prepared as described above or purchased from Ambion, Inc.(Austin, Tex.). Northern blotting was performed as described above(Example 14). The let-7c miRNA was observed to be expressed in spleen,kidney, testes, heart and liver tissues, as well as in HEK293 and T47Dcell lines. The let-7e miRNA was observed to be expressed in T-24, MCF7,T47D, 293T, HepG2, and K-562 cell lines.

In one embodiment, expression of let-7-like pri-miRNA homologs wasdetected in total RNA from brain, liver and spleen tissues, as well astotal RNA from preadipocytes, differentiated adipocytes, and HeLa,HEK-293, and T-24 cell lines by real-time RT-PCR. Primer/probe sets weredesigned to distinguish between and amplify specific let-7-likepri-miRNA homologs. These primer/probe sets are shown in Table 23.

TABLE 23 Primer/probe sets for assaying expression of let-7 miRNAhomologs Primer or Isis SEQ ID Pri-miRNA probe number NO. sequencelet-7b forward 341672 765 GAGGTAGTAGGTTGTGTGGTTTCA reverse 341673 766AGGGAAGGCAGTAGGTTGTATAGTT probe 341674 767 CAGTGATGTTGCCCCTCGGAAGAlet-7c forward 341675 768 TGCATCCGGGTTGAGGTA reverse 341676 769AGGAAAGCTAGAAGGTTGTACAGTTAA probe 341677 770AGGTTGTATGGTTTAGAGTTACACCCTGGGA let-7d forward 341678 771CCTAGGAAGAGGTAGTAGGTTGCA reverse 341679 772 CAGCAGGTCGTATAGTTACCTCCTTprobe 341680 773 AGTTTTAGGGCAGGGATTTTGCCCA let-7g forward 341681 774TTCCAGGCTGAGGTAGTAGTTTG reverse 341682 775 TTATCTCCTGTACCGGGTGGT probe341683 776 ACAGTTTGAGGGTCTAT let-7i forward 341684 777TGAGGTAGTAGTTTGTGCTGTTGGT reverse 341685 778 AGGCAGTAGCTTGCGCAGTTA probe341686 779 TTGTGACATTGCCCGCTGTGGAG let-7a-1 forward 341687 780GGATGAGGTAGTAGGTTGTATAGTTTTAGG reverse 341688 781CGTTAGGAAAGACAGTAGATTGTATAGTTATC probe 341689 782 TCACACCCACCACTGGlet-7a-3 forward 341690 783 GGGTGAGGTAGTAGGTTGTATAGTTTGG reverse 341691784 CACTTCAGGAAAGACAGTAGATTGTATAGTT probe 341692 785 CTCTGCCCTGCTATGG

Using these primer/probe sets, the let-7-like pri-miRNA homologs werefound to be expressed in human brain, liver and spleen, as well aspreadipocytes, differentiated adipocytes, and HeLa, T-24 and HEK-293cells lines. In particular, the let-7b pri-miRNA exhibited approximately100-fold higher expression in differentiated adipocytes as compared topreadipocytes. Furthermore, the let-7b, let-7c, let-7d, let-71, andlet-7a-3 pri-miRNAs were highly expressed in brain and spleen tissues.

In summary, the let-7-like homologs have been found to be widelyexpressed in various human tissues and several cell lines. Furthermore,some oligomeric compounds targeted to human let-7 pri-miRNAs generallyappeared to result in the induction of cell differentiation, consistentwith the functional role of let-7 as a regulator of developmental timingin nematode larva. Specifically, the oligomeric compounds targeted tolet-7c (ISIS Number 327931; SEQ ID NO: 349) and let-7a-3 (ISIS Number327903; SEQ ID NO: 321) resulted in an increase in expression levels forseveral markers of adipocyte differentiation. Furthermore, inhibition ofthe let-7-like homologs by oligomeric compounds of the present inventiondid not appear to induce caspases activated in apoptotic pathways(performed in Example 15). Thus, the oligomeric compounds of the presentinvention targeting let-7-like pri-miRNA homologs appear to stimulateadipocyte differentiation and do not promote cell death by apoptosis.Thus, the oligomeric compounds of the present invention may be useful aspharmaceutical agents in the treatment of anorexia or diseases,conditions or injuries in which the induction of cellulardifferentiation is desirable, such as Alzheimers disease or centralnervous system injury, in which neural regeneration would be beneficial.

Example 18 Effects of Oligomeric Compounds Targeting miRNAs on InsulinSignaling in HepG2 Cells

Insulin is secreted from pancreatic β-cells in response to increasingblood glucose levels. Through the regulation of protein expression,localization and activity, insulin ultimately stimulates conversion ofexcess glucose to glycogen, and results in the restoration of bloodglucose levels. Insulin is known to regulate the expression of over 100gene products in multiple cell types. For example, insulin completelyinhibits the expression of hepatic insulin-like growth factor bindingprotein-1 (IGFBP-1), a protein which can sequester insulin-like growthfactors, and phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-c) whichis a rate-controlling enzyme of hepatic gluconeogenesis. Levels of thefollistatin mRNA are also believed to decrease in response to insulintreatment. IGFBP-1 and PEPCK-c are overexpressed in diabetes, andPEPCK-c overexpression in animals promotes hyperglycemia, impairedglucose tolerance and insulin-resistance. Thus, the IGFBP-1, PEPCK-c andfollistatin genes serve as marker genes for which mRNA expression can bemonitored and used as an indicator of an insulin-resistant state.Oligomeric compounds with the ability to reduce expression of IGFBP-1,PEPCK-c and follistatin are highly desirable as agents potentiallyuseful in the treatment of diabetes and hypertension.

Oligomeric compounds of the present invention were tested for theireffects on insulin signaling in HepG2 cells. HepG2 cells were plated at7500 cells/well in collagen coated 96-well plates. The following day,cells were transfected with oligomeric compounds targeting miRNAs using100 nM oligomeric compound in LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) in two 96-well plates. The oligomeric compounds weretested in triplicate on each 96-well plate, except for positive andnegative controls, which were measured up to six times per plate. At theend of transfection, the transfection medium was replaced by regulargrowth medium. Twenty-eight hours post-transfection, the cells weresubjected to overnight (sixteen to eighteen hours) serum starvationusing serum free growth medium. Forty-four hours post-transfection, thecells in the transfected wells were treated with either no insulin(“basal” Experiment 1, for identification of insulin-mimetic compounds)or with 1 nM insulin (“insulin treated” Experiment 2, for identificationof insulin sensitizers) for four hours. At the same time, in bothplates, cells in some of the un-transfected control wells are treatedwith 100 nM insulin to determine maximal insulin response. At the end ofthe insulin or no-insulin treatment (forty-eight hourspost-transfection), total RNA is isolated from both the basal andinsulin treated (1 nM) 96-well plates, and the amount of total RNA fromeach sample is determined using a Ribogreen assay (Molecular Probes,Eugene, Oreg.). Real-time PCR is performed on all the total RNA samplesusing primer/probe sets for three insulin responsive genes: PEPCK-c,IGFBP-1 and follistatin. Expression levels for each gene are normalizedto total RNA, and values+standard deviation are expressed relative tothe transfectant only untreated control (UTC) and negative controlcompounds. Results of these experiments are shown in Tables 24 and 25.

TABLE 24 Experiment 1: Effects of oligomeric compounds targeting miRNAson insulin-repressed gene expression in HepG2 cells ISIS SEQ ID IGFBP-1PEPCK-c Follistatin Number NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTC N/AN/A 100 100 100 29848 737 N/A 95 87 94 n-mer 327876 294 mir-29b-1 93 119104 327878 296 mir-203 162 45 124 327880 298 mir-10b 137 110 107 327889307 mir-23b 56 137 56 327890 308 let-7I 99 85 78 327892 310mir-131-2/mir-9 108 75 91 327901 319 mir-143 133 119 93 327903 321let-7a-3 71 71 60 327905 323 mir-205 107 129 104 327913 331 mir-29c 123229 115 327919 337 mir-221 96 71 74 327922 340 mir-19b-2 109 77 57327925 343 mir-133b 152 145 110 327933 351 mir-145 125 118 112 327934352 mir-213 231 99 140 327939 357 mir-125b-1 125 125 104 327941 359mir-181b 83 101 80 327954 372 mir-148b 118 79 100 338664 491 mir-143 9075 93 pri-miRNA 340927 319 mir-143 201 87 111

Under “basal” conditions (without insulin), treatments of HepG2 cellswith oligomeric compounds of the present invention resulting indecreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/orfollistatin marker genes indicate that the oligomeric compounds have aninsulin mimetic effect. Treatments with oligomeric compounds of thepresent invention resulting in an increase in mRNA expression levels ofthe PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that thesecompounds inhibit or counteract the normal insulin repression of mRNAexpression of these genes.

From these data, it is evident that the oligomeric compounds, ISISNumber 327878 targeting mir-203 and ISIS Number 327922 targetingmir-19b-2, for example, result in a 55% and a 23% decrease,respectively, in PEPCK-c mRNA, a marker widely considered to beinsulin-responsive. Thus, these oligomeric compounds may be useful aspharmaceutic agents comprising insulin mimetic properties in thetreatment, amelioration, or prevention of diabetes or other metabolicdiseases.

Conversely, the results observed with the oligomeric compound targetingmir-29c (ISIS Number 327913), for example, exhibiting increasedexpression of the IGFBP-1, PEPCK-c and follistatin marker genes, suggestthat the mir-29c miRNA target may be involved in the regulation of theseinsulin-responsive genes. When the mir-29c miRNA is inactivated by anoligomeric compound, IGFBP-1, PEPCK-c and follistatin gene expression isno longer repressed.

TABLE 25 Experiment 2: Effects of oligomeric compounds targeting miRNAson insulin-sensitization of gene expression in HepG2 cells ISIS SEQ IDIGFBP-1 PEPCK-c Follistatin Number NO Pri-miRNA (% UTC) (% UTC) (% UTC)UTC(1 nm N/A N/A 100 100 100 insulin) 29848 737 N/A 92 94 97 n-mer327876 294 mir-29b-1 118 176 138 327878 296 mir-203 185 29 150 327880298 mir-10b 136 125 149 327890 307 let-7i 88 113 115 327892 308mir-131-2/ 139 104 96 mir-9 327901 310 mir-143 135 117 135 327903 319let-7a-3 81 87 89 327905 321 mir-205 115 147 148 327913 323 mir-29c 147268 123 327919 331 mir-221 154 105 178 327922 337 mir-19b-2 104 76 61327925 340 mir-133b 166 182 148 327933 343 mir-145 179 115 185 327934351 mir-213 244 105 103 327939 352 mir-125b-1 175 153 192 327941 357mir-181b 80 98 68 327954 359 mir-148b 120 102 105 327889 372 mir-23b 73202 72 338664 491 mir-143 100 76 84 pri-miRNA 340927 319 mir-143 285 103128

For HepG2 cells treated with 1 nM insulin, treatments with oligomericcompounds of the present invention resulting in a decrease in mRNAexpression levels of the PEPCK-c, IGFBP-1 and/or follistatin markergenes indicate that these compounds have an insulin sensitizationeffect. Treatments with oligomeric compounds of the present inventionresulting in an increase in mRNA expression levels of the PEPCK-c,IGFBP-1 and/or follistatin marker genes indicate that these compoundsinhibit or counteract the normal insulin response of repression of mRNAexpression of these genes.

From these data, it is evident that the oligomeric compounds, ISISNumber 327878 targeting mir-203 and ISIS Number 327922 targetingmir-19b-2, for example, were observed to result in a 71% and a 24%reduction, respectively, of PEPCK-c mRNA expression, widely consideredto be a marker of insulin-responsiveness. Thus, these oligomericcompounds may be useful as pharmaceutic agents with insulin-sensitizingproperties in the treatment, amelioration, or prevention of diabetes orother metabolic diseases.

Conversely, the results observed with the oligomeric compounds targetingmir-29c (ISIS Number 327913), mir-133b (ISIS Number 327925), andmir-125b-1 (ISIS Number 327939), all exhibiting increased expression ofthe IGFBP-1, PEPCK-c and follistatin marker genes, support theconclusion that the mir-29c, mir-133b, and mir-125b-1 miRNAs may beinvolved in the regulation of insulin-responsive genes. When thesemiRNAs are inactivated by the oligomeric compounds of the presentinvention, IGFBP-1, PEPCK-c and follistatin gene expression is no longerrepressed or insulin-sensitive.

A caspase assay was also performed (as in Example 11 above) in HepG2cells treated with oligomeric compounds of the present invention, and itwas determined that oligomeric compounds targeting the mir-29c,mir-133b, and mir-125b-1 miRNAs were not toxic to the cells and that theobserved reduction in mRNA expression levels of insulin-responsive geneswas not due to a general toxicity of the compounds or an induction ofapoptotic pathways.

Example 19 Analysis of Expression of mir-143 pri-miRNA and Maturemir-143

Ribonuclease Protection Assays:

The ribonuclease protection assay (RPA) is known in the art to be asensitive and accurate method of measuring and/or following temporalchanges in the expression of one or more RNA transcripts in a complexmixture of total RNA. Briefly, this method employs a radioactive probethat specifically hybridizes to a target transcript RNA. The probe isadded to a sample of total RNA isolated from tissues or cells ofinterest, and, upon hybridization to its target, the probe forms adouble-stranded RNA region. If the region of hybridization is shorterthan the entire length of either the probe or the target RNA molecule,the molecule will be a hybrid molecule with partial double-stranded andpartial single-stranded character. These hybrid molecules are thendigested with single-strand-specific RNases such as RNase A and/or T1,which remove any non-hybridized single stranded portions of the hybridmolecules, leaving only the “protected” dsRNA fragments. The RNaseprotected fragments are then electrophoresed on a denaturing gel,causing the strands to dissociate, and the intensity of radioactiveprobe signal observed is directly proportional to the amount of specifictarget transcript RNA in the original total RNA sample.

In an embodiment of the present invention, small non-coding RNAs in asample were detected by RPA using probes that hybridize to pri-miRNAs,pre-miRNAs or mature miRNAs. Probes were in vitro transcribed using themirVana™ miRNA Probe Construction Kit (Ambion Inc., Austin, Tex.)according to the manufacturer's protocol, beginning with a DNAoligonucleotide representing sense strand of the mature miRNA to bedetected plus four thymidylate residues plus an 8-base sequencecomplementary to the 3′-end of the T7 promoter primer supplied with thekit. When the T7 primer is annealed to this DNA oligonucleotide, theKlenow DNA polymerase is used to generate a double-stranded DNA, andthen in vitro transcription is performed using the T7 RNA polymerase andradiolabeled nucleotides to generate a radioactive RNA probe fordetection of the miRNA.

In one embodiment, a probe specifically hybridizing to the murinemir-143 miRNA was used in a RPA of 5 μg total RNA from kidney, liver,heart, lung, brain, spleen, and thymus tissues from mouse as well asadipose tissue from db/db obese mice, total RNA from an 11-day-oldembryo, and total RNA from undifferentiated and differentiated 3T3-L1cells. All signals were normalized to the levels of 5.8S rRNA.Expression levels of mir-143 were highest in lung, heart, spleen, thymusand kidney tissues from wildtype mice. Notably, mir-143 expressionlevels were significantly elevated in adipose tissue from db/db mice(approximately 4 times higher than expression levels in kidney, 2.4times higher than levels in heart and 1.6 times higher than levels inlung tissues from wildtype mice).

In one embodiment, a probe hybridizing to the mir-143 pri-miRNA moleculewas used in a RPA of 2-5 μg total RNA from human spleen, thymus, testes,heart, liver, kidney, skeletal muscle, brain, lung and adipose tissues,as well as total RNA from preadipocytes, differentiated adipocytes, andHepG2 cells. A probe hybridizing to the β-actin mRNA was used as acontrol. The highest levels of mir-143 pri-miRNA were observed in heart,kidney, thymus and adipose tissues, as well as in differentiatedadipocytes.

In one embodiment, a probe hybridizing to the mature mir-143 miRNA wasalso used in a RPA of 2 μg total RNA from human spleen, thymus, heart,liver, kidney and brain, tissues, as well as total RNA frompreadipocytes, differentiated adipocytes, and total RNA from HepG2,A549, T-24, HEK293, HuVEC (human umblical vein endothelial cells), HL-60and T47D cell lines. A probe hybridizing to the β-actin mRNA was used asa control, and all signals were normalized to the levels of mir-143expression in preadipocytes. The results are shown in Table 26.

TABLE 26 RNase protection of mature mir-143 in total RNA from tissuesand cell lines Tissue or cell line Fold Increase over preadipocytesSpleen 2.6 Thymus 3.8 Heart 8.2 Liver 0 Kidney 10.0 Brain 0.9Preadipocytes 1.0 Differentiated 2.6 adipocytes HepG2 0.5 A549 N.D. T-240.4 HEK293 0.5 HuVEC 0.3 HL-60 0.4 T47D 0.3

From these data, the highest levels of expression of the mature mir-143miRNA were observed in total RNA from kidney and heart tissues. Highlevels of expression of the mature mir-143 miRNA were also observed intotal RNA from lymphoid tissues such as spleen and thymus. Expression ofthe mature mir-143 miRNA is increased in differentiated adipocytes ascompared to levels in preadipocytes. These data also suggest that themir-143 miRNA plays a role in cellular differentiation.

In one embodiment, a uniform 2′-MOE phosphorothioate oligomeric compoundwith a sequence antisense to the mature mir-143 miRNA was spiked intothe RPA mixture above. This antisense mir-143 compound was found toblock the ribonuclease protection expression pattern previouslyobserved, suggesting that this antisense mir-143 oligomeric compoundspecifically hybridizes to and inhibits the activity of mir-143. Thisoligomeric compound targeting the mir-143 miRNA is predicted to form adouble stranded molecule that blocks endogenous mir-143 miRNA activitywhen employed in vivo.

It was also noted that, while expression of the mir-143 miRNA can bedetected in non-transformed cells, such as HuVECs, in general,transformed cell lines have not been observed to exhibit high levelsexpression of mir-143. When taken together with the observation that themir-143 miRNA is upregulated as adipocytes differentiate as well as theobservation that oligomeric compounds targeting mir-143 inhibitadipocyte differentiation, these data suggest that mir-143 normallypromotes adipocyte differentiation and mir-143 may have an inhibitoryeffect on cellular transformation that is consistent with its role inpromoting cellular differentiation. Lack of expression or downregulationof mir-143 in transformed cell lines may be a cause or consequence ofthe undifferentiated state. Thus, mir-143 mimics may be useful aspharmaceutical agents in the treatment of hyperproliferative disorderssuch as cancer.

In one embodiment, the expression of human mir-143 was assessed duringadipocyte differentiation. A probe hybridizing to the human mir-143miRNA was used in a RPA of 5 μg total RNA from pre-adipocytes, anddifferentiated adipocytes sampled at one, four, and ten dayspost-differentiation. All signals were normalized to the levels of 5.8SrRNA. mir-143 expression levels were 2.5 to 3-fold higher by day 10post-differentiation when compared to mir-143 expression levels inpre-adipocytes by ribonuclease protection assay.

Real-Time RT-PCR Analysis of mir-143 pri-miRNA Expression:

Expression levels of mir-143 pri-miRNA were compared in total RNAs fromvarious tissues and total RNA from several cell lines. Total RNA fromspleen, heart, liver, and brain tissues, as well as total RNA frompreadipocytes, differentiated adipocytes, and HepG2, T-24 and HeLa celllines was purchased or prepared as described supra. 80 ng of total RNAfrom each source was used to perform real-time RT-PCR using aprimer/probe set specific for the mir-143 pri-miRNA molecule. ISIS339314 (TCCCAGCCTGAGGTGCA; SEQ ID NO: 786) was used as the forwardprimer, ISIS 342897 (GCTTCATCTCAGACTCCCAACTG; SEQ ID NO: 787) was usedas the reverse primer, and ISIS 342898 (TGCTGCATCTCTG; SEQ ID NO: 788)was used as the probe. RNA levels from all sources were compared to RNAlevels from preadipocytes. Greater than 32-fold higher levels of mir-143pri-miRNA were observed in heart tissue as compared to preadipocytes;19-fold higher levels of mir-143 pri-miRNA were observed indifferentiated adipocytes relative to levels in preadipocytes; 5-foldhigher levels of mir-143 pri-miRNA were observed in spleen as comparedto preadipocytes.

Northern blot analyses were performed in differentiating adipocytes asdescribed in Example 14 using the mir-143-specific DNA oligonucleotideprobe (SEQ ID NO: 319) to detect the mir-143 target and a probe for theU6 RNA to normalize for variations in loading and transfer efficiency,and it was confirmed by Northern analysis that expression of maturemir-143 increases from day 1 through day 10 after induction ofdifferentiation.

In human pre-adipocytes and adipocytes sampled one, four, seven and tendays post-differentiation, expression levels of mir-143 pri-miRNA werealso assessed using real-time RT-PCR analysis as described herein. 80 ngof total RNA from pre-adipocytes or differentiated adipocytes was usedto perform real-time RT-PCR using the same primer/probe set specific forthe mir-143 pri-miRNA molecule described supra (ISIS 339314, SEQ ID NO:786 was used as the forward primer, ISIS 342897, SEQ ID NO: 787 was usedas the reverse primer, and ISIS 342898, SEQ ID NO: 788 was used as theprobe). RNA levels from all sources were normalized to 5.8S rRNA levels.mir-143 pri-miRNA levels in preadipocytes were 94% of the level of the5.8S rRNA. At day 1 post-differentiation, mir-143 pri-miRNA levels haddecreased to 38% of the level of the 5.8S rRNA. By day 4post-differentiation, mir-143 pri-miRNA levels had decreased to 26%, byday 7 post-differentiation, mir-143 pri-miRNA levels were at 25%, and byday 10 post-differentiation, mir-143 pri-miRNA levels had dropped to 23%of the level of the 5.8S rRNA. Taken together with the results from RPAanalysis, it appears that levels of the mature mir-143 miRNA increasesapproximately 2- to 3-fold by day 10 post-differentiation indifferentiated adipocytes, accompanied by a concomittant approximately4-fold decrease in the levels of unprocessed mir-143 pri-miRNA,indicating that adipocyte differentiation coincides with either anincrease in processing of the mir-143 miRNA from the mir-143 pri-miRNAor an overall decrease in mir-143 pri-miRNA production.

Effects of Oligomeric Compounds on Expression of pri-miRNAs:

Mature miRNAs originate from long endogenous primary transcripts(pri-miRNAs) that are often hundreds of nucleotides in length. It isbelieved that a nuclear enzyme in the RNase III family, known as Drosha,processes pri-miRNAs (which can range in size from about 110 nucleotidesup to about 450 nucleotides in length) into pre-miRNAs (from about 70 to110 nucleotides in length) which are subsequently exported from thenucleus to the cytoplasm, where the pre-miRNAs are processed by humanDicer into double-stranded intermediates resembling siRNAs, which arethen processed into mature miRNAs. Using the real-time RT-PCR methodsdescribed herein, the expression levels of several pri-miRNAs werecompared in differentiating adipocytes. Total RNA from preadipocytes anddifferentiating adipocytes was prepared as described herein.

In one embodiment, modified oligomeric compounds can be transfected intopreadipocytes or other undifferentiated cells, which are then induced todifferentiate (as described in detail, herein), and it can be determinedwhether these modified oligomeric compounds act to inhibit or promotecellular differentiation. Real-time RT-PCR methods can then be used todetermine whether modified oligomeric compounds targeting miRNAs canaffect the expression or processing of the pre-miRNAs from the pri-miRNA(by the Drosha enzyme), the processing of the mature miRNAs from thepre-miRNA molecules (by the Dicer enzyme), or the RISC-mediated bindingof a miRNA to its target nucleic acid.

Here, oligomeric compounds targeting mir-143 were transfected intopreadipocytes which were then induced to differentiate, in order toassess the effects of these compounds on mir-143 pri-miRNA levels duringdifferentiation. mir-143 pri-miRNA levels were assessed on days 3 and 9after differentiation.

In addition to the uniform 2′-MOE phosphorothioate oligomeric compoundISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143, a 5-10-7gapmer oligomeric compound, ISIS Number 340927 (SEQ ID NO: 319), wasdesigned to target mature mir-143. As negative controls, “scrambled”oligomeric compounds were also designed; ISIS Number 342672(ATACCGCGATCAGTGCATCTTT; incorporated herein as SEQ ID NO: 789) contains13 mismatches with respect to the mature mir-143 miRNA, and ISIS Number342673 (SEQ ID NO: 758) contains 15 mismatches with respect to themature mir-143 miRNA. ISIS 342672 and ISIS 342673 are uniform 2′-MOEphosphorothioate oligomeric compounds 22 nucleotides in length. ISISNumber 342677 (SEQ ID NO: 789) and ISIS Number 342678 (SEQ ID NO: 758)are the corresponding 5-10-7 scrambled 2′-MOE gapmer oligomericcompounds. All cytidine residues are 5-methylcytidines. Additionally,ISIS Number 342683 (CCTTCCCTGAAGGTTCCTCCTT; herein incorporated as SEQID NO: 790), representing the scrambled sequence of an unrelated PTP1Bantisense oligonucleotide, was also used as a negative control.

These compounds were transfected into differentiating adipocytes andtheir effects on levels of the mir-143 pri-miRNA molecule were assessedin pre-adipocytes vs. differentiated adipocytes, by real-time RT-PCRusing the primer/probe set specific for the mir-143 pri-miRNA (forwardprimer=ISIS 339314, SEQ ID NO: 786; reverse primer=ISIS 342897, SEQ IDNO.: 787; probe=ISIS 342898, SEQ ID NO.: 788). Thus, it was observedthat in the presence of the oligomeric compound ISIS Number 327901 (SEQID NO: 319), levels of the mir-143 pri-miRNA are enhanced approximately4-fold in differentiated adipocytes 9 days post-differentiation ascompared to 3 days post-differentiation. These results suggest that ISISNumber 327901, the uniform 2′-MOE P═S oligomeric compound targeted tomature mir-143, interferes with the processing of the mir-143 pri-miRNAinto the pre-miRNA by the Drosha RNase III enzyme. Alternatively, thecompound interferes with the processing of the mir-143 pre-miRNA intothe mature mir-143 miRNA by the Dicer enzyme. The decrease in levels ofmature mir-143 miRNA in differentiating cells treated with ISIS Number327901 (SEQ ID NO: 319) may also trigger a feedback mechanism thatsignals these cells to increase production of the mir-143 pri-miRNAmolecule. Not mutually exclusive with the processing interference or thefeedback mechanisms is the possibility that treatment with oligomericcompounds could stimulate the activity of an RNA-dependent RNApolymerase (RdRP) that amplifies the mir-143 pri-miRNA or pre-miRNAmolecules. Oligomeric compounds of the present invention are predictedto disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinderDrosha and/or Dicer cleavage, respectively. Furthermore, oligomericcompounds which are capable of binding to the mature miRNA are alsopredicted to prevent the RISC-mediated binding of a miRNA to its targetnucleic acid, either by cleavage or steric occlusion of the miRNA.

Example 20 Identification of RNA Transcripts Bound by miRNAs

The RACE-PCR method (Rapid Amplification of cDNA Ends) was used as ameans of identifying candidate RNA transcripts bound and/or potentiallyregulated by miRNAs. RNA was prepared and isolated from preadipocytes,and, using the SMART RACE cDNA Amplification kit (BD Biosciences,Clontech, Palo Alto, Calif.) according to manufacturer's protocol,synthetic adaptor sequences were incorporated into both the 5′- and3′-ends of the amplified cDNAs during first strand cDNA synthesis. 5′RACE-PCR was then performed using the mature miRNA as the 3′-end primeralong with the 5′ adapter primer from the kit to amplify the 5′-end ofthe candidate RNA transcript. 3′ RACE-PCR was performed using theantisense sequence of the miRNA as a primer along with the 3′ adapterprimer from the kit to amplify the 3′-end of the candidate RNAtranscript. In some embodiments, the primers 2-nucleotides shorter thanthe corresponding miRNA were used in order to identify targets with somemismatching nucleotides at the end of the miRNA (these primers areindicated by “3′-RACE-2 nt” in Table 27 below).

For example, the antisense sequences of the mature mir-43, let-7g,mir-23b, mir-29c, mir-131, mir-143, mir-130b and mir-213 miRNAs wereused as primers in 3′ RACE-PCR, and the mature mir-143 or mir-15asequences were used in 5′ RACE-PCR. The RACE-PCR products employing themir-143 miRNA, the mir-143 antisense sequence, the mir-131 antisensesequence or the mir-15a miRNA as primers were electrophoresed and gelpurified, prominent bands were excised from the gel, and these productswere subcloned using standard laboratory methods. The subcloned productsfrom the RACE-PCR were then were sent to Retrogen, Inc. (San Diego,Calif.) for sequencing. Candidate RNA transcripts targeted by miRNAswere thereby identified.

Candidate RNA targets identified by RACE-PCR methods are shown in Table27, where the miRNA-specific primer used to identify each transcript isindicated in the column entitled “primer”. (In some cases, the targetwas identified multiple times by more than one RACE-PCR method, and thusappears in the table more than once).

TABLE 27 Predicted RNA targets of mir-143 SEQ GenBank ID Primer MethodAccession RNA transcript targeted by miRNA NO mir-143 5′RACE NM_001753.2caveolin 1, caveolae protein, 22 kDa 791 mir-143 5′RACE NM_004652.1ubiquitin specific protease 9, X- 792 linked (fat facets-like,Drosophila) mir-143 5′RACE NM_007126.2 valosin-containing protein 793mir-143 5′RACE NM_000031.1 aminolevulinate, delta-, dehydratase 794mir-143 5′RACE NM_007158.1 NRAS-related gene 795 mir-143 5′RACENM_015396.1 HSPC056 protein 796 mir-143 5′RACE NM_001219.2 calumenin 797mir-143 5′RACE BC051889.1 RNA binding motif, single stranded 798interacting protein 1 mir-143 5′RACE BX647603.1 Homo sapiens mRNA; cDNA799 DKFZp686L01105 (from clone DKFZp686L01105) mir-143 5′RACE AB051447.1KIAA1660 protein 800 mir-143 5′RACE NM_007222.1 zinc-fingers andhomeoboxes 1 801 mir-143 5′RACE NM_001855.1 collagen, type XV, alpha 1802 mir-143 3′RACE NM_007222.1 zinc-fingers and homeoboxes 1 801 mir-1433′RACE NM_006732 FBJ murine osteosarcoma viral 803 oncogene homolog Bmir-143 3′RACE NM_003718.2 cell division cycle 2-like 5 804(cholinesterase-related cell division controller) mir-143 3′RACENM_005626.3 splicing factor, arginine/serine- 805 rich 4 mir-143 3′RACENM_002355.1 mannose-6-phosphate receptor (cation 806 dependent) mir-1433′RACE NM_000100.1 cystatin B (stefin B) 807 mir-143 3′RACE NM_015959.1CGI-31 protein 808 mir-143 3′RACE NM_006769.2 LIM domain only 4 809mir-143 3′RACE NM_003184.1 TAF2 RNA polymerase II, TATA box 810 bindingprotein (TBP)-associated factor, 150 kDa mir-143 3′RACE NM_025107.1 myctarget in myeloid cells 1 811 mir-143 3′RACE NM_003113.1 nuclear antigenSp100 812 mir-143 3′RACE NM_002696.1 polymerase (RNA) II (DNA directed)813 polypeptide G mir-143 3′RACE NM_004156.1 protein phosphatase 2(formerly 2A), 814 catalytic subunit, beta isoform mir-143 3′RACENM_031157 heterogeneous nuclear 815 ribonucleoprotein A1 mir-143 3′RACENM_004999.1 myosin VI 817 mir-143 3′RACE NM_018036.1 chromosome 14 openreading frame 103 818 mir-143 3′RACE NM_018312.2 chromosome 11 openreading frame 23 819 mir-143 3′RACE NM_002950.1 ribophorin I 820 mir-1433′RACE NM_006708.1 glyoxalase I 821 mir-143 3′RACE NM_014953.1 mitoticcontrol protein dis3 homolog 822 mir-143 3′RACE NM_004926.1 zinc fingerprotein 36, C3H type- 823 like 1 mir-143 3′RACE NM_004530.1 matrixmetalloproteinase 2 824 (gelatinase A, 72 kDa gelatinase, 72 kDa type IVcollagenase) mir-143 3′RACE NM_015208.1 KIAA0874 protein 825 mir-1433′RACE NM_002582.1 poly(A)-specific ribonuclease 826 (deadenylationnuclease) mir-143 3′RACE NM_000297.2 polycystic kidney disease 2 827(autosomal dominant) mir-143 3′RACE NM_001175 Rho GDP dissociationinhibitor (GDI) 828 beta mir-143 3′RACE XM_166529 glucocorticoid inducedtranscript 1, 837 GLCCI1 mir-143 3′RACE- NM_001753.2 caveolin 1,caveolae protein, 22 kDa 791 2nt mir-143 3′RACE- NM_006732 FBJ murineosteosarcoma viral 803 2nt oncogene homolog B mir-143 3′RACE-NM_000100.1 cystatin B (stefin B) 807 2nt mir-143 3′RACE- NM_015959.1CGI-31 protein 808 2nt mir-143 3′RACE- NM_004156.1 protein phosphatase 2(formerly 2A), 814 2nt catalytic subunit, beta isoform mir-143 3′RACE-NM_031157 heterogeneous nuclear 815 2nt ribonucleoprotein A1 mir-1433′RACE- NM_002582.1 poly(A)-specific ribonuclease 826 2nt (deadenylationnuclease) mir-143 3′RACE- NM_000297.2 polycystic kidney disease 2 8272nt (autosomal dominant) mir-143 3′RACE- NM_006325.2 RAN, member RASoncogene family 829 2nt mir-143 3′RACE- NM_004627.1 tryptophan richbasic protein 830 2nt mir-143 3′RACE- NM_012210.1 tripartitemotif-containing 32 831 2nt mir-143 3′RACE- AJ131244.1 SEC24 relatedgene family, member A 832 2nt (S. cerevisiae) mir-143 3′RACE-NM_031267.1 cell division cycle 2-like 5 833 2nt (cholinesterase-relatedcell division controller) mir-143 3′RACE- AL049367.1 guanine nucleotidebinding protein 835 2nt (G protein), gamma 12 mir-143 3′RACE- NM_001344defender against cell death 1 836 2nt mir-131 3′RACE AK001214.1hypothetical protein FLJ10352 1735 mir-131 3′RACE NM_001614 actin, gamma1 (ACTG1), mRNA 1736 mir-131 3′RACE NM_001948.1 dUTP pyrophosphatase(DUT), mRNA 1737 mir-131 3′RACE NM_002387.1 mutated in colorectalcancers (MCC), 1738 mRNA mir-131 3′RACE NM_004109.1 ferredoxin 1 (FDX1),nuclear gene 1739 encoding mitochondrial protein, mRNA mir-131 3′RACENM_004342.4 caldesmon 1 (CALD1), transcript 1740 variant 2, mRNA mir-1313′RACE NM_005572.2 lamin A/C (LMNA), transcript variant 1741 2, mRNAmir-131 3′RACE NM_015640.1 PAI-1 mRNA-binding protein (PAI- 1742 RBP1),mRNA mir-131 3′RACE NM_017789.1 semaphorin 4C (SEMA4C), mRNA 1743mir-131 3′RACE NM_144697.1 hypothetical protein BC017397 1744(LOC148523), mRNA mir-131 3′RACE NM_173710 NADH dehydrogenase 3 (MTND3),mRNA 1745 mir-15a 5′RACE AF220018.1 Homo sapiens tripartite motif 1746protein (TRIM2) mRNA mir-15a 5′RACE M98399.1 Human antigen CD36 mRNA1747 mir-15a 5′RACE Y00281.1 Human mRNA for ribophorin I 1748

Because these RNA transcripts in Table 27 were identified as being boundby one of the mir-143, mir-131, or mir-15a miRNAs, these miRNAs arepredicted to serve a regulatory role in expression or activity of thesetranscripts identified by RACE-PCR. Additional candidate human RNAtargets can be identified in the same manner.

Example 21 Effects of Oligomeric Compounds on Adipocyte DifferentiationHallmark Genes in Differentiated Adipocytes

The effect of the oligomeric compounds of the present inventiontargeting miRNAs on the expression of markers of cellulardifferentiation was examined in differentiated adipocytes.

The effects of the oligomeric compounds of the present invention on thehallmark genes known to be upregulated during adipocyte differentiationassayed in Example 13 were also assayed in differentiated adipoctyes. Aspreviously described, the HSL, aP2, Glut4, and PPARγ genes playimportant rolls in the uptake of glucose and the metabolism andutilization of fats. Also as previously described, an increase intriglyceride content is another well-established marker for adipocytedifferentiation. Human white preadipocytes (Zen-Bio Inc., ResearchTriangle Park, N.C.) were grown in preadipocyte media (ZenBio Inc.).After the cells reached confluence (approximately three days), they wereexposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-γagonist, IBMX, dexamethasone, and insulin for three days. Cells werethen fed Adipocyte Medium (Zen-Bio, Inc.), which was replaced at 2 to 3day intervals. One day before transfection, 96-well plates were seededwith 3000 cells/well. Cells were then transfected on day ninepost-differentiation, according to standard published procedures with250 nM oligonucleotide in LIPOFECTIN™ (Invitrogen Corporation, Carlsbad,Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomericcompounds were tested in triplicate on each 96-well plate, and theeffect of TNF-α, known to inhibit adipocyte differentiation, was alsomeasured in triplicate. Oligomeric compound treatments andtransfectant-only negative controls may be measured up to six times perplate. On day twelve post-differentiation, cells were washed and lysedat room temperature, and the expression of the four hallmark genes, HSL,aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation weremeasured in adipocytes transfected with the uniform 2′-MOEphosphorothioate (PS) previously described in Example 13 as well as thechimeric gapmer oligomeric compounds targeting the mir-143 miRNA and themir-143 pri-miRNA described in Example 16. On day twelvepost-differentiation, cells were lysed in a guanadinium-containingbuffer and total RNA was harvested. The amount of total RNA in eachsample was determined using a Ribogreen Assay (Molecular Probes, Eugene,Oreg.). Real-time PCR was performed on the total RNA using primer/probesets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2,and PPARγ. Triglyceride levels as well as mRNA levels for each of thefour adipocyte differentiation hallmark genes are expressed relative tocontrol levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). Theresults of this experiment are shown in Table 28.

TABLE 28 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers ISIS SEQ ID Number NO TG aP2 HSLGlut4 PPAR gamma 327876 294 1.16 0.67 0.81 3.53 1.28 327878 296 1.080.13 0.19 0.17 0.85 327880 298 1.12 1.14 0.93 0.76 1.86 327888 306 1.130.73 0.84 0.56 1.69 327889 307 1.09 1.12 0.77 0.99 1.63 327890 308 1.130.35 0.42 0.37 1.05 327892 310 1.23 0.81 0.62 0.42 1.01 327901 319 1.121.28 1.47 2.20 1.34 327903 321 1.12 0.56 0.53 0.36 0.91 327905 323 1.180.85 0.65 0.58 1.31 327913 331 1.12 1.05 1.09 1.52 1.29 327919 337 1.151.20 0.83 1.82 1.80 327922 340 1.48 0.91 1.01 0.61 0.99 327925 343 1.330.78 1.20 0.74 1.30 327933 351 1.63 1.58 1.30 2.12 1.60 327934 352 1.431.50 1.97 1.52 1.54 327939 357 1.33 1.16 1.08 0.72 1.89 327941 359 1.330.90 1.17 0.90 1.66 327954 372 1.46 1.23 1.35 0.61 1.46 328382 491 1.330.92 0.53 0.75 0.97 338664 491 1.72 0.77 1.01 1.08 1.06 340927 319 1.610.71 0.64 0.96 1.21

From these data, it was observed that the compound targeting the mir-203miRNA (ISIS Number 327878), exhibited a sustained reduction in thehallmark marker genes at the 12^(th) day post differentiation. Treatmentwith this compound resulted in decreased expression of the aP2, HSL,Glut4 and PPARγ marker genes, indicating that this oligomeric compoundmay lead to reduced levels of mobilization of fatty acids from adiposetissue, and has the potential to ameliorate some of the symptoms of type2 diabetes, obesity, hypertension, atherosclerosis, cardiovasculardisease, insulin resistance, and certain cancers. Notably, the effect oftreatment of differentiated adipocytes with this oligomeric compoundtargeting the mir-203 miRNA mirrors the effect of treating cells withthe TNF-α positive control that inhibits adipocyte differentiation. Thisevidence suggests that the oligomeric compound targeting the mir-203miRNA can act as a TNF-α mimetic compound, and potentially may be usedin the suppression of cellular differentiation and the maintenance ofcells in a quiescent state.

The oligomeric compound targeting the mir-203 miRNA was also tested inthe insulin assay (see Example 18) and was observed to reduce expressionof PEPCK-c, indicating that it may also be useful as an insulin mimeticand/or antidiabetic drug.

As an extension of these conclusions, one having ordinary skill in theart would appreciate that further modified oligomeric compounds could bedesigned to also target the mir-203 mature miRNA, or the pri-miRNA andpre-miRNA precursors. Such compounds are noted to be within the scope ofthe present invention.

Example 22 Effects of Oligomeric Compounds on Lymphocytic Leukemia Cells

Mir15-a-1 and mir-16-3 have been recently shown to reside in humanchromosomal region (13q14) that is deleted in about 50% of chroniclymphocytic leukemia (CLL) patients. Mir-15 and 16 were found to bedown-regulated in about 68% of CLL cases (Calin et al., Proc. Natl.Acad. Sci. USA, 2002, 99, 15524-15529, which is incorporated herein byreference in its entirety). CLL B-cells develop chemotherapy resistanceover time, possibly due to a defective apoptosis pathway.

Using the 5′RACE method (described in Example 20), the CD36 mRNA wasidentified as one target regulated by mir-15 and/or mir-16 miRNAs. CD36is a scavenger receptor involved in fat uptake by macrophages andadipocytes. CD36 is reported to be upregulated in some CLL cell lines,and its expression may correlate with tumor invasiveness.

If the apoptosis pathway is defective and the deletion ordown-regulation of mir-15 and/or mir-16 play a role in CLLchemo-resistance, then addition of mir-15 and/or mir-16 should be ableto induce apoptosis in CLL and increase drug-induced apoptosis. RNAoligonucleotide molecules ISIS Number 338963 (TAGCAGCACATAATGGTTTGTG;SEQ ID NO: 269) representing mir-15a-1/mir-15a-2, ISIS Number 338961(TAGCAGCACATCATGGTTTACA; SEQ ID NO: 246) representing mir-15b, and ISISNumber 338965 (TAGCAGCACGTAAATATTGGCG; SEQ ID NO: 196) representingmir-16-1/mir-16-2/mir-16-3 were synthesized and deprotected.Additionally, RNA oligonucleotides bearing imperfect complementarity tothese miRNA mimics (mimicking the imperfect complementarity found in thepri-miRNA) were also synthesized and deprotected. These imperfectcomplements were ISIS Number 338964 (TGCAGGCCATATTGTGCTGCCT; SEQ ID NO:840), which is partially complementary to ISIS Number 338963 andrepresents the imperfect complement of mir-15a-1/mir-15a-2; ISIS Number338962 (TGCGAATCATTATTTGCTGCTC; SEQ ID NO: 841), which is partiallycomplementary to ISIS Number 338961 and represents the imperfectcomplement of mir-15b; ISIS Number 338966 (CTCCAGTATTAACTGTGCTGCTG; SEQID NO: 842), which is partially complementary to ISIS Number 338965 andrepresents the imperfect complement of mir-16-1 and mir-16-2; and ISISNumber 338967 (CACCAATATTACTGTGCTGCTT; SEQ ID NO: 843), which ispartially complementary to ISIS Number 338965 and represents theimperfect complement of mir-16-3. These RNA molecules were diluted inwater, and the concentration determined by A₂₆₀. Equimolar amounts ofeach of the miRNAs and their imperfect complementary RNA sequences weremixed together in the presence of Dharmacon 5× Universal buffer to formfour “natural” double-stranded miRNA mimics. ISIS Number 338965 (SEQ IDNO: 196) was used twice; once, it was hybridized to ISIS Number 338966,and once it was hybridized to ISIS Number 338967, to form two different“natural” double-stranded miRNA mimics, Mir-16-1/Mir-16-2 and Mir-16-3,with imperfect complementarity. The mixture of four “natural” miRNAmimics was incubated for 1-5 minutes at 90° C. (the time depends on thevolume of the mixture) and then incubated at 37° C. for one hour. A₂₆₀readings were taken on the mixture for final concentrationdetermination.

Heparinized peripheral blood from CLL patients was separated on a Ficolldensity gradient to obtain greater than 95% pure CLL B-cells. Thesecells are tested for expression of the CD5/CD19/CD23 antigens. Positiveexpression of these three antigens indicates that the cells are CLLB-cells (Pederson et al., Blood, 2002, 100, 2965, which is incorporatedherein by reference in its entirety). Additionally, cytogenetic analysiscan be performed to ascertain that the cells have the 13q deletion. Amixture of all four “natural” miRNA mimics at 2 μM each waselectroporated into the cells. The cells were cultured in the presenceor absence of apoptosis-inducing agents fludarabine A, or Dexamethasone(which are known to employ the intrinsic mitochondrial apoptoticpathway) or the antitumor agent CDDO-Im (reported to function through analternative extrinsic apoptotic pathway) for 24 hours. Followingincubation, apoptosis was monitored by annexin/PI double staining asoutlined in FIG. 1 of Pederson et al., Blood, 2002, 100, 2965. Thedouble-stranded RNA oligomeric compounds representing mir-15 and mir-16miRNA mimics were observed to play a role in the induction ofspontaneous as well as drug-induced apoptosis. Thus, oligomericcompounds of the present invention may be useful in the treatment ofCD36-related diseases and conditions such as chronic lymphocyticleukemia and other cancers.

Example 23 Effect of Oligomeric Compounds Targeting miRNAs in vivo

As described herein, leptin-deficient (ob/ob) mice, leptinreceptor-deficient (db/db) mice and diet-induced obesity (DIO) mice areused to model obesity and diabetes. In accordance with the presentinvention, oligomeric compounds targeting mir-143, mir-131 (also knownas mir-9) and mir-203 were tested in the ob/ob and db/db models. Theob/ob mice were fed a high fat diet and were subcutaneously injectedwith the oligomeric compounds of the invention or a control compound ata dose of 25 mg/kg two times per week for 6 weeks. Saline-injectedanimals, leptin wildtype littermates (i.e. lean littermates) and ob/obmice fed a standard rodent diet served as controls. The physiologicaleffects resulting from inhibition of target RNA, such as the effects oftarget inhibition on glucose and insulin metabolism and the expressionof genes that participate in lipid metabolism, cholesterol biosynthesis,fatty acid oxidation, fatty acid storage, gluconeogenesis and glucosemetabolism, were assessed by methods disclosed herein. In brief, plasmalevels of liver transaminases, cholesterol, triglycerides, free fattyacids and glucose were assessed weekly by tail bleed, with the tailbleed on week three taken under fasting conditions. After the treatmentperiod, mice were sacrificed and liver, spleen, pancreas, muscle, kidneyand heart, as well as brown adipose tissue (BAT) and white adiposetissue (WAT) tissues were collected. mRNA expression levels of theGlut4, aP2, HSL and PPARγ marker genes were evaluated. RNA isolation andtarget RNA expression level quantitation are performed as described.

Two oligomeric compounds targeting the mir-143 miRNA were compared fortheir effects on the physiological indications of obesity and diabetes.The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319),22-nucleotides in length, targets the mature mir-143, and is a uniform2′-MOE oligonucleotide with phosphorothioate internucleoside linkagesthroughout. The oligomeric compound ISIS Number 340927 (SEQ ID NO: 319)is a 5-10-7 gapmer also designed to target the mature mir-143 miRNA. Theeffects of these oligomeric compounds targeting mir-143 on severalphysiological parameters and markers of obesity and/or diabetes wereexamined in vivo. Potential effects on food consumption were alsomonitored.

Plasma cholesterol levels were observed to slightly decrease over timein ob/ob mice treated with the gapmer oligomeric compound ISIS Number340927 (SEQ ID NO: 319) targeted to mir-143. Similarly, plasmatriglyceride and plasma glucose levels were generally slightly lower inob/ob mice treated with this compound as compared to untreated mice, ormice treated with control compounds. mRNA expression levels of theGlut4, aP2 and HSL marker genes were slightly reduced by both oligomericcompounds ISIS Number 327901 and ISIS Number 340927 targeting mir-143.Thus, these oligomeric compounds targeting mir-143 may be usefulcompounds in the treatment of obesity or diabetes.

In addition, Northern blot analyses were performed to quantitate theexpression of mature mir-143 in kidney samples of ob/ob mice treatedwith oligomeric compounds of the present invention. The mir-143 specificDNA oligonucleotide probe (SEQ ID NO: 319) described above was used todetect expression levels of the mir-143 miRNA in ob/ob mice treated(twice weekly at 25 mg/kg) with ISIS Numbers 327901, the uniform 2′-MOEoligomeric compound, or ISIS Number 340927, the 5-10-7 gapmer compound,both targeted to mir-143, versus saline treated animals or animalstreated with ISIS 342672 (SEQ ID NO: 789), a uniform 2′-MOE scramblednegative control oligomeric compound. Expression levels were normalizedagainst the U6 RNA and the expression levels of saline treated animalswere set at 100%. Most notably, in kidney samples from ob/ob micetreated with ISIS Number 327901, the uniform 2′-MOE oligomeric compoundtargeted to mir-143 exhibited a nearly 40% decrease in in vivoexpression levels of the mature mir-143 miRNA. In kidney samples frommice treated with the gapmer oligomeric compound targeting mir-143, ISISNumber 340927, a 23% reduction in in vivo expression levels of themature mir-143 miRNA was observed.

Oligomeric compounds targeting the mir-131/mir-9 and the mir-203 miRNAswere also tested for their effects on the physiological indicators ormarkers of obesity and diabetes. The oligomeric compound, ISIS Number327892 (SEQ ID NO: 310), targeted to mir-131/mir-9,21-nucleotides inlength, is a uniform 2′-MOE oligonucleotide with phosphorothioateinternucleoside linkages throughout. The oligomeric compound ISIS Number340926 (SEQ ID NO: 310) is a 5-10-6 gapmer oligomeric compound alsodesigned to target the mir-131/mir-9 miRNA. The oligomeric compound ISISNumber 327878 (SEQ ID NO: 296) targeted to mir-203, 22-nucleotides inlength, is a uniform 2′-MOE oligonucleotide with phosphorothioateinternucleoside linkages throughout. The oligomeric compound ISIS Number345349 (SEQ ID NO: 296) is a 5-10-7 gapmer oligomeric compound alsodesigned to target the mir-203 miRNA. The effects of these oligomericcompounds were examined in vivo in the ob/ob model. Potential effects onfood consumption were also monitored.

Fed plasma glucose levels in ob/ob mice treated with the oligomericcompounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926(SEQ ID NO: 310) targeted to mir-131/mir-9, and ISIS Number 327878 (SEQID NO: 296) and ISIS Number 345349 (SEQ ID NO: 296) targeted to mir-203were observed to be reduced beginning at approximately four weeks afterthe start of treatment and continuing to decrease on week five ascompared to untreated mice, or mice treated with control compounds.Triglyceride levels were also observed to be reduced over time in micetreated with ISIS 340926 and 345349, the gapmer oligomeric compoundstargeted to mir-131/mir-9 and mir-203, respectively. No signs of livertoxicity were indicated by weekly measurements of plasma transaminasesupon treatment of ob/ob mice with any of the oligomeric compoundstargeting mir-143, mir-203 or mir-131/mir9.

ob/ob mice in the fasted state on day 19 after treatment with theoligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 also exhibitedsignificant reductions in plasma glucose levels. Notably, the gapmeroligomeric compound ISIS Number 340926 (SEQ ID NO: 310) targeted tomir-131/mir-9 was even more potent than the corresponding uniform 2′-MOEoligonucleotide ISIS Number 327892 (SEQ ID NO: 310).

Furthermore, a decrease in food consumption was observed by the thirdweek and this reduced level was maintained in the fourth weekpost-treatment of ob/ob mice with these oligomeric compounds. Therefore,the oligomeric compounds targeting the mir-131/mir-9 and mir-203 miRNAshave potential use as appetite suppressants, as well as in the treatmentof obesity or diabetes.

The oligomeric compounds ISIS Number 327901 and ISIS Number 340927 bothtargeting mir-143, ISIS Number 327892 and ISIS Number 340926 bothtargeting mir-131/mir-9, and ISIS Number 327878 and ISIS Number 345349both targeting mir-203 were also tested in db/db mice. Althoughtreatment of db/db mice with the gapmer compounds targeting mir-143,mir-203 or mir-131/mir9 resulted in an approximately 2-fold increase inliver transaminases in db/db mice, the uniform 2′-MOE oligomericcompounds targeting mir-143, mir-203 or mir-131/mir-9 were not found tocause liver toxicity in db/db mice, as assessed by weekly measurementsof plasma transaminase levels.

Additional oligomeric compounds targeting miRNAs were studied in ob/obmice. Six week old ob/ob mice were treated (dose=25 mg/kg, twice weeklyfor four weeks) with uniform 2′-MOE and gapmer oligomeric compoundstargeting mir-143, mir-23b, mir-221, let-7a, and mir-29b, and comparedto saline treated animals or animals treated with ISIS 342672 (SEQ IDNO: 789), a uniform 2′-MOE scrambled negative control oligomericcompound bearing 13 base mismatches to mir-143. Expression levels werenormalized against the U6 RNA and the expression levels of salinetreated animals were set at 100%. Fed plasma samples were takenbi-weekly by tail bleed, and plasma levels of liver transaminases,cholesterol, triglycerides, free fatty acids and glucose were assessed,with the tail bleed on week three taken under fasting conditions. Ob/obmice were treated with ISIS Numbers 327901 and 340927, the uniform2′-MOE and gapmer oligomeric compounds, respectively, targeting mir-143are described above. Additionally, ob/ob mice were also treated with thefollowing compounds: ISIS Number 327889 (SEQ ID NO: 307), aphosphorothioate uniform 2′-MOE oligomeric compound, and ISIS Number340925 (SEQ ID NO: 307), a 2′-MOE 5-10-8 gapmer oligomeric compound,each targeting mir-23b; ISIS Number 327919 (SEQ ID NO: 337), a uniform2′-MOE oligomeric compound, and ISIS Number 345384 (SEQ ID NO: 337), aphosphorothioate 2′-MOE 5-10-8 gapmer oligomeric compound, eachtargeting mir-221; ISIS Number 327903 (SEQ ID NO: 321), a uniform 2′-MOEoligomeric compound, and ISIS Number 345370 (SEQ ID NO: 321), aphosphorothioate 2′-MOE 5-10-7 gapmer oligomeric compound, eachtargeting let-7a; and ISIS Number 327876 (SEQ ID NO: 294), a uniform2′-MOE oligomeric compound, and ISIS Number 345347 (SEQ ID NO: 294), aphosphorothioate 2′-MOE 5-10-8 gapmer oligomeric compound, each targetedto mir-29b-1.

Ob/ob mice treated with the gapmer compounds ISIS 340925 and ISIS345384, targeting mir-23b and mir-221, respectively, exhibitedreductions in plasma glucose levels in the fed state at weeks two andfour, as compared to untreated mice, or mice treated with controlcompounds. Furthermore, mice treated with ISIS 340925 exhibited adecrease in triglycerides in the fourth week. Ob/ob mice treated withISIS 340925 did not exhibit an increase in plasma transaminases at weekstwo or four. Thus, the oligomeric compounds ISIS Numbers 340925 and345384 may be useful as agents for the treatment of obesity and/ordiabetes.

In addition, Northern blot analyses were performed to quantitate theexpression of mir-23b in kidney samples of ob/ob mice treated witholigomeric compounds of the present invention. To detect the mir-23btarget, a target-specific DNA oligonucleotide probe with the sequenceGTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) was synthesized by IDT(Coralville, Iowa). The oligo probes were 5′ end-labeled with T4polynucleotide kinase with (γ-³²P) ATP (Promega). The mir-23b specificDNA oligonucleotide probe was used to detect expression levels of themir-23b miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISISNumbers 327889, the uniform 2′-MOE oligomeric compound, or ISIS Number340925, the 5-10-8 gapmer compound, both targeted to mir-23b, versussaline treated animals or animals treated with a control oligomericcompound, ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), auniform 5-10-52′-MOE gapmer targeting an unrelated gene, PTEN.Expression levels were normalized against the U6 RNA and the expressionlevels of saline treated animals were set at 100%. Most notably, inkidney samples from ob/ob mice treated with ISIS Number 327889, theuniform 2′-MOE oligomeric compound targeted to mir-23b exhibited anearly 64% decrease in in vivo expression levels of the mir-23b miRNA.In kidney samples from mice treated with the gapmer oligomeric compoundtargeting mir-23b, ISIS Number 340925, a 41% reduction in in vivoexpression levels of the mir-23b miRNA was observed.

As described, supra, the C57BL/6 mouse strain is reported to besusceptible to hyperlipidemia-induced atherosclerotic plaque formation,and when these mice are fed a high-fat diet, they develop diet-inducedobesity (DIO). Accordingly, the DIO mouse model is useful for theinvestigation of obesity and development of agents designed to treatthese conditions. In one embodiment of the present invention, oligomericcompounds targeting miRNAs were tested in the DIO model. Normal C57/BL6male mice were fed a high fat diet (40% fat, 41% carbohydrate, 18%protein) for 12 weeks before the study began. DIO mice were thenrandomized by weight and insulin values. Initial body fat compositionwas determined by Dual X-ray Absorptiometry (DEXA) Scan. DIO mice werethen subcutaneously injected with oligomeric compounds of the inventionat a dose of 25 mg/kg, twice weekly. DIO mice were treated witholigomeric compounds ISIS Numbers 327901 and 340927 targeting mir-143,ISIS Numbers 327892 and 340926 targeting mir-131/mir-9, ISIS Numbers327878 and ISIS Number 345349 targeting mir-203, and ISIS Numbers 327889and 340925, targeting mir-23b. As negative controls, “scrambled”oligomeric compounds were also designed: ISIS Number 342672 contains 13mismatches with respect to the mature mir-143 miRNA; ISIS Number 353607(ACTAGTTTTTCTTACGTCTGA; herein incorporated as SEQ ID NO: 845) is aphosphorothioate 5-10-62′-MOE gapmer oligomeric compound containing 12mismatches with respect to mir-131/mir-9; ISIS Number 353608(CTAGACATTAGCTTTGACATCC; herein incorporated as SEQ ID NO: 846) is aphosphorothioate 5-10-72′-MOE gapmer oligomeric compound containing 16mismatches with respect to mir-203. DEXA scans were also performed atweeks 0, 3 and 5 after treatment with the oligomeric compounds to assessthe fat mass to lean mass ratio. The effects of target inhibition onlevels of plasma glucose and insulin, liver transaminases, cholesteroland triglycerides, were also assessed weekly by tail bleed, and afterthe treatment period, mice were sacrificed and liver and kidney heart,as well as white adipose tissue (WAT) tissues collected. The mRNAexpression levels of the Glut4, aP2, HSL and PPARγ marker genes are alsoassessed. Treatment of DIO mice with the uniform 2′-MOE oligomericcompounds ISIS 327901 targeting mir-143, ISIS 327892 targetingmir-131/mir9, ISIS 327878 targeting mir-203, and ISIS 327889 targetingmir-23b did not appear to cause liver toxicity in these mice as assessedby weekly measurements of plasma transaminase levels. Similarly, thegapmer oligomeric compounds ISIS 340927 targeting mir-143, and ISIS340926 targeting mir-131/mir-9, 340925 did not cause significantincreases in liver toxicity, and the gapmer compound ISIS 340925targeting mir-23b caused only an approximately 2-fold increase in theliver transaminase AST. Interestingly, the gapmer compounds ISIS Numbers340927 targeting mir-143, 340926 targeting mir-131/mir-9, 345349targeting mir-203, and 340925, targeting mir-23b were all effective atreducing insulin levels at the two and four week time points, ascompared to saline-treated control mice. Furthermore, some improvementin body composition (a reduction in body weight and fat mass) wasobserved. These data from the DIO model suggest that oligomericcompounds targeting mir-143, mir-131/mir-9, mir-203 and mir-23b may beuseful as agents for the treatment of obesity and/or diabetes.

Having the information disclosed herein, one of ordinary skill in theart would comprehend that of other classes of inhibitors targetingmir-143, mir-209, mir-131/mir-9 and mir-23b miRNAs, such as antibodies,small molecules, and inhibitory peptides, can be assessed for theireffects on the physiological indicators of diseases in in vivo models,and these inhibitors can be developed for the treatment, amelioration orimprovement of physiological conditions associated with a particulardisease state or condition. Such inhibitors are envisioned as within thescope of the instant invention.

Example 24 Effects of Oligomeric Compounds on Cell Cycling

Cell Cycle Assay:

Cell cycle regulation is the basis for various cancer therapeutics. Cellcycle checkpoints are responsible for surveillance of proper completionof certain steps in cell division such as chromosome replication,spindle microtubule attachment and chromosome segregation, and it isbelieved that checkpoint functions are compromised in some cancerouscells. Furthermore, because the shift from quiescence to an activelygrowing state as well as the passage through mitotic checkpoints areessential transitions in cancer cells, most current chemotherapy agentstarget dividing cells. For example, by blocking the synthesis of new DNArequired for cell division, an anticancer drug can block cells inS-phase of the cell cycle. These chemotherapy agents impact many healthyorgans as well as tumors. In some cases, a cell cycle regulator willcause apoptosis in cancer cells, but allow normal cells to undergogrowth arrest and therefore remain unaffected. Loss of tumor supressorssuch as p53 sensitizes cells to certain anticancer drugs; however,cancer cells often escape apoptosis. Further disruption of cell cyclecheckpoints in cancer cells can increase sensitivity to chemotherapywhile allowing normal cells to take refuge in G1 and remain unaffected.A goal of these assays is to determine the effects of oligomericcompounds on the distribution of cells in various phases of the cellcycle.

In some embodiments, the effects of several oligomeric compounds of thepresent invention were examined in the normal human foreskin fibroblastBJ cell line, the mouse melanoma cell line B16-F10 (also known as B16cells), as well as the breast carcinoma cell line, T47D. These celllines can be obtained from the American Type Culture Collection(Manassas, Va.). BJ cells were routinely cultured in MEM high glucosewith 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodiumbicarbonate and supplemented with 10% fetal bovine serum, 0.1 mMnon-essential amino acids and 1.0 mM sodium pyruvate (all media andsupplements from Invitrogen Life Technologies, Carlsbad, Calif.).B16-F10 cells were routinely cultured in DMEM high glucose (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). T47D cells werecultured in DMEM High glucose media (Invitrogen Life Technologies,Carlsbad, Calif.) supplemented with 10% fetal bovine serum. Cells wereroutinely passaged by trypsinization and dilution when they reached 80to 90% confluence. Cells were plated on collagen-coated 24-well plates(Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately50,000 cells per well and allowed to attach to wells overnight.

As a negative control, a random-mer oligomeric compound, 20 nucleotidesin length, ISIS 29848 (SEQ ID NO: 737) was used. In addition, a positivecontrol, ISIS 183891 (CCGAGCTCTCTTATCAACAG; herein incorporated as SEQID NO: 847) was included; ISIS 183891 targets kinesin-like 1 (also knownas Eg5) and inhibits cell cycle progression. Eg5 is known to induceapoptosis when inhibited. ISIS 29248 and ISIS 183891 are chimericoligomeric compounds (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”(a “5-10-5 gapmer”). The wings are composed of 2′-methoxyethoxy (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the compound. All cytidine residuesare 5-methylcytidines. ISIS 340348 (CTACCTGCACGAACAGCACTTT; hereinincorporated as SEQ ID NO: 848) is a uniform 2′-MOE phosphorothioateoligomeric compound targeting the mir-93 miRNA, and ISIS 340365(TACTTTATATAGAACACAAG; herein incorporated as SEQ ID NO: 849) is a5-10-5 gapmer phosphorothioate oligomeric compound targeting themir-92-2 miRNA.

Oligomeric compounds were mixed with LIPOFECTIN™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve a final concentration of 150nM of oligomeric compound and 4.5 μg/ml LIPOFECTIN™. Before adding tocells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ were mixedthoroughly and incubated for 0.5 hrs. The medium was removed from theplates and each well was washed in 250 μl of phosphate-buffered saline.The wash buffer in each well was replaced with 250 μL of the oligomericcompound/OPTI-MEM™/LIPOFECTIN cocktail. Control cells receivedLIPOFECTIN™only. The plates were incubated for 4 hours at 37° C., afterwhich the medium was removed. 100 μl of full growth medium was added toeach well. After 72 hours, routine procedures were used to prepare cellsfor flow cytometry analysis and cells were fixed with ethanol andstained with propidium iodide to generate a cell cycle profile using aflow cytometer. The cell cycle profile was analyzed with the ModFitprogram (Verity Software House, Inc., Topsham Me.).

Fragmentation of nuclear DNA is a hallmark of apoptosis and produces anincrease in cells with a hypodiploid DNA content. Cells with ahypodiploid DNA content are categorized as “subG1.” The cells in the G1,G2/M and S phases are considered to be cycling, and cells in the subG1and aneuploid categories are considered to have left the cell cycle. Anincrease in cells in G1 phase is indicative of a cell cycle arrest priorto entry into S phase; an increase in cells in S phase is indicative ofcell cycle arrest during DNA synthesis; and an increase in cells in theG2/M phase is indicative of cell cycle arrest just prior to or duringmitosis. Data are shown in Table 29 and expressed as percentage of cellsin each phase of the cell cycle.

TABLE 29 Effects of oligomeric compounds targeting miRNAs on cellcycling SEQ ISIS # ID # Pri-miRNA SubG1 G1 S G2/M aneuploid UTC N/A N/A8.1 59.6 27.5 12.9 7.3 ISIS- 737 N/A 9.6 57.8 26.5 15.6 12 29848 n-merISIS- 847 Kinesin- 20.8 33.1 39.2 27.6 11.5 183891 like 1/Eg5 Positivecontrol 327878 296 mir-203 17.3 39.1 40.8 20 11.9 327888 306 mir-108-113.3 53.7 29.5 16.7 12.9 327889 307 mir-23b 8.2 53.1 32.5 14.4 10.5327901 319 mir-143 12 34.7 44.9 20.3 13.6 327902 320 mir-192-1 10.6 50.733.9 15.3 13.4 327903 321 let-7a-3 11 53.7 30.9 15.4 13.4 327904 322mir-181a 8.6 54.4 29.5 16.2 15.6 327905 323 mir-205 8.5 56.9 28.1 1514.7 327906 324 mir-103-1 15.2 46.1 33 20.9 15.8 327907 325 mir-26a 17.849.5 32.8 17.6 17.8 327908 326 mir-33a 5.6 55.4 29.2 15.3 13.1 327909327 mir-196-2 7.9 52.6 30.1 17.3 16.3 327910 328 mir-107 9.3 49.5 3317.5 13.1 327911 329 mir-106 10.9 49.9 30.1 20 16.5 327914 332 mir-130a8.5 55.8 28.9 15.3 16.2 327919 337 mir-221 10.8 54.3 30.3 15.4 16 327922340 mir-19b-2 10 50.4 30.7 18.9 16.8 327928 346 mir-29a-1 6.6 56 27.9 1615.9 327933 351 mir-145 10.2 49.6 31.3 19.1 15.9 327934 352 mir-213 6.654.4 28.2 17.4 17 327941 359 mir-181b 8.2 57.2 29.9 12.9 15.8 327951 369mir-15a-1 4.3 60.9 24.8 14.3 16.7 328342 451 mir-203 4.8 62.3 24.9 12.815.2 328362 471 mir-108-1 9.1 51.2 33.6 15.1 12.9 328364 473 mir-23b 1.961.5 24.2 14.3 15.1 328382 491 mir-143 2.9 59.8 25.7 14.4 14.8 328388497 let-7a-3 4.0 57.5 28 14.6 14.5 328394 503 mir-181a 2.4 59.5 24.5 1618.3 328396 505 mir-205 4.6 56.8 28.2 15 19.8 328419 528 mir-221 6.051.2 32.5 16.3 17.9 328423 532 mir-19b-2 4.9 52.9 32.4 14.8 15.3 328424533 mir-19b-2 3.1 61.9 23.7 14.4 16.9 328436 545 mir-29a-1 3.5 59.2 26.913.9 17.4 328644 553 mir-145 7.2 58.4 27.6 14 17.5 328691 600 mir-1457.7 60.5 24.4 15.1 16.6 328697 606 mir-181b 2.4 57.6 26.4 16 13.5 328773682 mir-15a-2 2.7 56.4 26.9 16.7 11.7 340348 848 mir-93 14.1 53.9 31.814.3 12.3 340365 849 mir-92-2 4.3 55.2 29.4 15.4 18.3

From these data, it is evident that treatment with the oligomericcompounds targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319);mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number327906 (SEQ ID NO: 324); mir-106, ISIS Number 327911 (SEQ ID NO: 329);and mir-145, ISIS Number 327933 (SEQ ID NO: 351) resulted in anincreased percentage of cells in the G2/M phase, indicating that theseoligomeric compounds arrest or delay the cell cycle at or just prior tomitosis, potentially activating a mitotic checkpoint.

Treatment with the oligomeric compounds targeting mir-26a, ISIS Number327907 (SEQ ID NO: 325); mir-205, ISIS Number 328396 (SEQ ID NO: 505);mir-181a, ISIS Number 328394 (SEQ ID NO: 503); and mir-92-2, ISIS Number340365 (SEQ ID NO: 849) resulted in higher than average percentages ofaneuploid cells, indicating that these oligomeric compounds interferewith proper chromosome segregation.

Treatment with the oligomeric compounds targeting mir-203, ISIS Number327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324);mir-26a, ISIS Number 327907 (SEQ ID NO: 325); and mir-93, ISIS Number340348 (SEQ ID NO: 848) resulted in an increased percentage of cellswith hypodiploid DNA content (SubGl phase) indicating that theoligomeric compound treatment may induce apoptotic events.

The effects of several oligomeric compounds of the present inventionwere also examined in the HeLa and A549 human carcinoma cell lines, bothof which can be obtained from the American Type Culture Collection(Manassas, Va.).

In some embodiments, HeLa cells were plated on collagen-coated 24-wellplates at 50,000-60,000 cells per well, and allowed to attach to wellsovernight. In some embodiments, HeLa cells were synchronized by doublethymidine block (cells were washed three times with PBS, then grown in10% FBS containing 2 mM thymidine; then 19 hours later, cells werewashed three times in PBS, 10% FBS for 9 hours; cells were thenincubated in 10% FBS, 2 mM thymidine for 15 hours; then washed threetimes with PBS, 10% FBS and samples were taken every two hours over a 16hour period). A portion of each time sample was fixed with ethanol andtreated with propidium iodide and subjected to FACs analysis fordetermination of the percentage of cells in each phase of the cellcycle. Distinctive peaks were observed for G0-, S-, Early G2/M-, LateG2/M-, and G1-phases of the cell cycle at 0-, 4-, 6-, 8-, and 12-hours,respectively, indicating that the cells were synchronized. HeLa cellstreated with 10 μM cisplatin or 100 ng/ml nocodazole were used ascontrols for G1-phase and late G2/M-phases, respectively. From theremaining portion of each of these time samples, total RNA was isolatedand used to assess the expression of cell cycle marker mRNAs using thereal-time RT-PCR methods and/or used to screen microarrays to assess theexpression of miRNAs over the course of the cell cycle. It was observedthat several miRNAs are expressed in a cell-cycle-dependent manner.Shown in Table 30 are the mRNA levels of the E2F1 transcription factorand topoisomerase 2A (Top2A), which vary over the course of the cellcycle and can be used for comparison to the experimental groups for theconfirmation of cell cycle phase. Data are an average of three trials.

TABLE 30 Expression levels of cell cycle markers treatment E2F1 mRNATop2A mRNA  10 uM cisplatin 102 15 100 ng/ml nocodazole 23 176  0 hrs(G0-phase) 100 100  4 hrs (S-phase) 81 105  6 hrs (early G2/M-phase) 39221  8 hrs (late G2/M-phase) 50 254  12 hrs (G1-phase) 61 124

In some embodiments, HeLa cells were also treated with oligomericcompounds targeting miRNAs. As described above, oligomeric compoundswere mixed with LIPOFECTIN™ in OPTI-MEM™ (Invitrogen Life Technologies,Carlsbad, Calif.) to a final concentration of 150 nM of oligomericcompound and 6 μg/ml LIPOFECTIN™. Before adding to cells, the oligomericcompound, LIPOFECTIN™ and OPTI-MEM™ were mixed thoroughly and incubatedfor 0.5 hrs. The medium was removed from the plates. Each well waswashed in 250 μl of PBS. The wash buffer in each well was replaced with250 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN cocktail. Controlcells received LIPOFECTIN™ only. The plates were incubated for 4 hoursat 37° C., after which the medium was removed. 1000 μl of full growthmedium was added to each well. After 24 hours (Table 31) or 48 hours(Table 32), cells were prepared for flow cytometry analysis to generatea cell cycle profile. The cell cycle profile was analyzed with theModFit program (Verity Software House, Inc., Topsham Me.).

The random-mer ISIS 29848 (SEQ ID NO: 737) was used as a negativecontrol, and ISIS 183891 (SEQ ID NO: 847), targeting kinesin-like 1/Eg5,was included as a positive control. Results of these experiments areshown in Tables 31 and 32. Data are expressed as percentage of cells ineach phase relative to the untreated control (UTC); values above 100 areconsidered to indicate a delay or arrest in that phase of the cellcycle. Table 31 shows the results from cells sampled 24 hours afteroligomeric compound treatment, and Table 32 shows the results from cellssampled 48 hours after oligomeric compound treatment. In some cases, thesame oligomeric compound was tested in repeated experiments.

TABLE 31 Effects of oligomeric compounds targeting miRNAs on cellcycling (24 hours) % cells in cell cycle phase Pri-miRNA ISIS # SEQ ID #subG1 G1 S G2/M aneuploid UTC N/A N/A 100 100 100 100 100 n-mer  29848737 120 116 81 108 76 Kinesin-like 1/Eg5 183891 847 251 21 109 231 95collagen, type I, 338797 624 197 101 79 148 193 alpha 1/hypotheticalmiRNA-144 hypothetical miRNA-039 338666 493 235 123 63 158 102hypothetical miRNA-111 328111 413 62 127 75 99 50 hypothetical miRNA-111338750 577 107 148 76 97 166 hypothetical miRNA-142 328115 417 177 90 87147 59 hypothetical miRNA-154 328119 421 75 100 94 112 83 hypotheticalmiRNA-154 328724 633 155 91 90 135 197 hypothetical miRNA-179 328749 658312 126 82 110 138 hypothetical miRNA-179 328780 689 124 96 87 136 149hypothetical miRNA-181 328136 438 330 125 81 88 51 hypotheticalmiRNA-181 338833 660 232 150 56 142 185 let-7a-3 327903 321 118 92 104106 98 let-7a-3 328388 375 120 110 83 115 85 mir-100-1 327957 497 197 9188 145 66 mir-100-1 328707 616 188 36 93 195 166 mir-103-1 327906 324228 153 47 107 65 mir-103-1 328397 506 134 93 86 142 91 mir-106 327911329 158 130 62 122 104 mir-106 328403 512 284 70 85 197 53 mir-106328403 512 189 86 75 179 82 mir-107 327910 328 174 154 42 118 73mir-108-1 328362 471 114 101 87 126 66 mir-10a 327949 367 194 82 84 17268 MiR-125a, Mouse 341787 852 221 113 75 144 165 mir-127, Mouse 341788853 303 154 54 140 114 mir-130b 328687 596 231 80 98 131 149 mir-130b338769 596 188 171 61 103 133 mir-131-2/mir-9 327892 310 153 86 111 10380 mir-131-2/mir-9 328369 310 84 100 88 125 71 mir-131-2/mir-9 340926478 286 98 91 121 83 mir-133b 338713 540 93 152 72 101 187 mir-141338741 568 157 141 73 112 166 mir-143 327901 319 108 101 94 110 90mir-143 328382 491 81 118 76 116 78 mir-143 328382 491 226 102 80 144202 mir-143 340927 319 118 121 75 111 88 mir-143 340927 319 131 128 71106 87 mir-145 327933 351 192 102 83 131 92 mir-145 327933 351 190 90 91140 47 mir-145 328644 553 71 113 84 109 68 mir-145 345395 351 247 54 82222 77 mir-149, Mouse 341785 854 125 152 92 53 158 mir-152 328727 636245 133 81 105 161 mir-152 338809 636 106 159 82 69 210 mir-16-3 327877295 154 107 66 159 62 mir-17/mir-91 327885 303 151 129 63 121 55mir-181a-1 327904 322 114 99 102 99 89 mir-182 328744 653 229 31 108 167111 mir-182 338826 653 145 148 79 90 138 mir-192-1 327902 320 178 57 106176 66 mir-192-1 327902 320 175 44 121 163 98 mir-192-1 328383 492 31455 82 222 92 mir-192-1 328383 492 289 63 97 183 98 mir-192-1 338665 340173 85 76 175 193 mir-19b-2 327922 492 131 97 96 114 104 mir-19b-2328424 533 60 110 85 112 74 mir-203 327878 296 124 96 94 122 73 mir-203328342 451 192 33 95 238 67 mir-205 327905 323 144 99 88 129 50 mir-205327905 323 149 94 95 121 98 mir-205 328396 505 97 94 87 139 88 mir-205338678 505 162 122 75 131 202 mir-211 327946 364 225 90 84 156 43mir-211 328674 583 564 125 93 84 69 mir-211 338756 583 137 147 75 99 166mir-213/mir-181a-2 327934 352 278 87 85 160 55 mir-213/mir-181a-2 327934352 204 118 66 137 77 mir-213/mir-181a-2 328647 556 140 101 92 119 140mir-216 327956 374 120 124 68 120 61 mir-216 328759 668 239 88 78 168184 mir-22 327896 314 121 83 103 128 65 mir-22 328374 483 198 54 115 16297 mir-220 327944 362 165 85 110 111 50 mir-221 327919 337 85 92 103 10996 mir-221 328419 528 87 109 79 124 77 mir-23a 338836 663 153 185 53 105150 mir-23b 327889 307 122 104 102 87 82 mir-23b 340925 307 151 103 89117 73 mir-26a-1 327907 325 224 119 77 111 75 mir-26a-1 345373 325 19666 94 176 68 mir-29b-1 327876 294 103 98 104 95 66 mir-29b-1 327876 294149 93 92 131 75 mir-29b-1 328337 446 107 106 88 113 104 mir-29b-1328337 446 99 108 88 109 64 mir-29b-2 328339 448 235 77 102 143 61mir-29c 338690 517 149 124 78 123 194 mir-30a 328084 585 381 43 104 163101 mir-30b 328676 585 139 99 86 134 169 mir-30b 338758 743 113 129 81108 190 mir-30d 328421 530 288 47 105 200 70 mir-33a 327908 326 138 9899 106 114 mir-92-1 327897 315 143 114 80 115 69 mir-92-1 327897 315 180128 74 100 54 mir-92-2 340365 849 109 125 71 114 84 mir-95 (Mourelatos)340350 855 218 183 54 104 94 mir-96 338637 464 88 170 70 84 188

TABLE 32 Effects of oligomeric compounds targeting miRNAs on cellcycling (48 hours) % cells in cell cycle phase Pri-miRNA ISIS # SEQ ID #subG1 G1 S G2/M aneuploid UTC N/A N/A 100 100 100 100 100 n-mer  29848737 86 87 121 117 109 Kinesin-like 1/Eg5 183891 847 173 19 124 331 72collagen, type I, 338797 624 813 66 124 168 175 alpha 1/hypotheticalmiRNA-144 hypothetical miRNA-039 338666 493 1832 44 136 217 125hypothetical miRNA-111 328111 413 371 84 126 119 90 hypotheticalmiRNA-111 338750 577 201 99 101 103 190 hypothetical miRNA-142 328115417 195 92 114 107 86 hypothetical miRNA-154 328119 421 767 75 145 12481 hypothetical miRNA-154 328724 633 653 70 134 140 155 hypotheticalmiRNA-179 328749 658 962 37 129 246 65 hypothetical miRNA-179 328780 689917 83 130 110 133 hypothetical miRNA-181 328136 438 140 83 133 113 85hypothetical miRNA-181 338833 660 1091 44 106 258 154 let-7a-3 327903321 74 102 95 98 94 let-7a-3 328388 375 112 99 101 102 126 mir-100-1327957 497 864 65 169 127 85 mir-100-1 328707 616 1486 46 134 213 155mir-103-1 327906 324 57 100 98 103 83 mir-103-1 328397 506 74 97 101 10996 mir-106 327911 329 65 99 96 109 101 mir-106 328403 512 863 61 177 13185 mir-106 328403 512 108 82 148 106 80 mir-107 327910 328 53 99 91 11192 mir-108-1 328362 471 87 96 104 108 97 mir-10a 327949 367 773 66 157138 71 MiR-125a, Mouse 341787 852 707 55 126 197 153 mir-127, Mouse341788 853 748 76 105 163 116 mir-130b 328687 596 1119 55 174 131 171mir-130b 338769 596 482 76 116 149 194 mir-131-2/mir-9 327892 310 121 74150 129 79 mir-131-2/mir-9 328369 310 72 99 95 109 109 mir-131-2/mir-9340926 478 68 83 120 131 106 mir-133b 338713 540 426 95 104 109 194mir-141 338741 568 185 100 101 99 170 mir-143 327901 319 93 98 104 103104 mir-143 328382 491 71 102 92 103 109 mir-143 328382 491 350 83 122120 133 mir-143 340927 319 95 91 107 121 113 mir-143 340927 319 83 91107 122 108 mir-145 327933 351 91 76 135 138 86 mir-145 327933 351 43880 133 123 75 mir-145 328644 553 52 101 101 98 82 mir-145 345395 351 21351 192 157 87 mir-149, Mouse 341785 854 1148 82 126 116 166 mir-152328727 636 846 68 152 124 140 mir-152 338809 636 345 86 110 129 157mir-16-3 327877 295 755 59 152 168 80 mir-17/mir-91 327885 303 456 78129 133 76 mir-181a-1 327904 322 116 87 126 114 80 mir-182 328744 6531774 31 78 334 171 mir-182 338826 653 696 61 124 182 137 mir-192-1327902 320 1176 39 171 208 81 mir-192-1 327902 320 202 44 166 205 87mir-192-1 328383 492 303 53 217 124 90 mir-192-1 328383 492 940 54 178150 90 mir-192-1 338665 340 1629 40 89 292 149 mir-19b-2 327922 492 8196 105 109 91 mir-19b-2 328424 533 89 103 91 101 111 mir-203 327878 29650 89 119 114 92 mir-203 328342 451 189 55 115 225 107 mir-205 327905323 719 48 194 150 67 mir-205 327905 323 100 78 143 122 99 mir-205328396 505 88 89 114 119 129 mir-205 338678 505 1158 81 78 188 179mir-211 327946 364 431 72 150 129 76 mir-211 328674 583 1663 69 160 109134 mir-211 338756 583 311 90 121 100 169 mir-213/mir-181a-2 327934 352752 62 156 152 92 mir-213/mir-181a-2 327934 352 155 66 148 155 117mir-213/mir-181a-2 328647 556 589 69 153 118 136 mir-216 327956 374 18491 106 121 110 mir-216 328759 668 1744 50 31 343 148 mir-22 327896 314886 55 140 194 66 mir-22 328374 483 787 65 157 143 71 mir-220 327944 362490 75 129 144 78 mir-221 327919 337 104 80 122 139 104 mir-221 328419528 83 99 96 107 112 mir-23a 338836 663 811 52 152 169 165 mir-23b327889 307 133 78 137 130 101 mir-23b 340925 307 89 87 130 109 93mir-26a-1 327907 325 116 92 111 115 94 mir-26a-1 345373 325 116 75 132145 119 mir-29b-1 327876 294 41 87 120 119 100 mir-29b-1 327876 294 25176 141 126 69 mir-29b-1 328337 446 66 92 105 119 108 mir-29b-1 328337446 662 73 143 135 74 mir-29b-2 328339 448 678 73 153 123 92 mir-29c338690 517 413 91 110 112 190 mir-30a 328084 585 1028 20 168 241 57mir-30b 328676 585 366 86 118 118 172 mir-30b 338758 743 267 103 99 92153 mir-30d 328421 530 1103 30 202 198 64 mir-33a 327908 326 61 99 98105 93 mir-92-1 327897 315 134 100 103 95 84 mir-92-1 327897 315 125 94114 105 63 mir-92-2 340365 849 71 99 94 109 129 mir-95 (Mourelatos)340350 855 1144 76 126 134 125 mir-96 338637 464 239 90 109 117 210

Several oligomeric compounds were observed to result in an arrest ordelay of the cell cycle, in some cases correlating with acell-cycle-dependent expression profile as determined by miRNAmicroarray analysis.

For example, from these data, it was observed that treatment of HeLacells with oligomeric compounds (MOE-gapmers and fully modified MOEs)targeting miRNAs caused an increase in the percentage of cellsexhibiting a subG1-phase or aneuploid DNA content, indicating aberrantchromosome segregation. Treatment with oligomeric compounds ISIS Number338797 (SEQ ID NO: 624) targeted to hypothetical miRNA-144, ISIS Number338833 (SEQ ID NO: 660) targeted to hypothetical miRNA-181, and ISISNumber 328759 (SEQ ID NO: 668) targeted to mir-216, each appeared tocause an induce chromosome missegregation events at both the 24-hour and48-hour timepoints. Thus, these compounds may be useful in triggering acheckpoint arrest in rapidly dividing cells, potentially useful in thetreatment of hyperproliferative disorders such as cancer.

It was also observed that other oligomeric compounds (MOE-gapmers andfully modified MOEs) targeting miRNAs appeared to induce an arrest ordelay in the G1-, S-, or G2/M-phases of the cell cycle. By miRNAmicroarray analysis, expression levels of the mir-205 miRNA wereobserved to increase in the S- and G1-phases of the cell cycle in HeLacells. Treatment of HeLa cells with the oligomeric compound ISIS Number327905 (SEQ ID NO: 323), targeting the mir-205 miRNA, was observed toarrest or delay the cell cycle in S-phase at the 48-hour time point,suggesting that the mir-205 miRNA may play a role in regulating DNAreplication. It was also observed that treatment of HeLa cells with theoligomeric compound ISIS Number 338678 (SEQ ID NO: 505), targeted to themir-205 pri-miRNA, resulted in an arrest or delay primarily in theG2/M-phase of the cell cycle, suggesting that this oligomeric compoundmay interfere with processing of the miRNA precursor into a maturemiRNA, which appears to have an impact on mitosis.

Treatment of HeLa cells with oligomeric compounds ISIS Number 327892(SEQ ID NO: 310), targeting mir-131/mir-9, and ISIS Number 327934 (SEQID NO: 352), targeting mir-213/mir-181a-2, was observed to arrest ordelay the cell cycle in G2/M- and S-phases at the 48-hour time point,suggesting that the mir-131/mir-9 and mir-213/mir-181a-2 miRNAs areinvolved in regulating DNA replication and entry into mitosis.

Treatment of HeLa cells with oligomeric compound ISIS Number 345373 (SEQID NO: 325), targeting miR-26a-1, was observed to arrest or delay cellsmainly in the G2/M-phase at 24 hrs after oligonucleotide-treatment, andat 48 hrs after oligonucleotide-treatment to arrest or delay cellsmainly in S-phase of the cell cycle, suggesting that miR-26a-1 isinvolved in mitosis and that cells making it through a first round ofcell division may harbor errors that cause them to arrest during a newround of DNA replication.

By miRNA microarray analysis, expression levels of the mir-145 miRNAwere observed to increase in the G2/M-phase of the cell cycle in HeLacells, and treatment of HeLa cells with the oligomeric compounds ISISNumber 327933 (SEQ ID NO: 351), a uniform 2′-MOE compound, and ISISNumber 345395 (SEQ ID NO: 351), a chimeric 2′-MOE gapmer compound, bothtargeting the mir-145 miRNA, were observed to arrest or delay the cellcycle in G2/M-phase at the 24-hour time point and at subG1-phase at the48-hour time point, suggesting that the mir-145 miRNA plays a role inmitosis and that cells making it through a first round of cell divisionmay harbor errors that cause them to arrest before a new round of DNAreplication.

By miRNA microarray analysis, expression levels of the mir-192-1 miRNAwere observed to increase in the G2/M-phase of the cell cycle in HeLacells, and treatment of HeLa cells with the oligomeric compounds ISISNumber 327902 (SEQ ID NO: 320), a uniform 2′-MOE compound, and ISISNumber 328383 (SEQ ID NO: 492), a chimeric 2′-MOE gapmer compound,targeted against the mir-192-1 miRNA and the mir-192-1 precursor,respectively, were observed to arrest or delay the cell cycle in theG2/M-phase at 24-hours after oligonucleotide treatment, and at both theS- and G2/M-phases at the 48-hour time point, suggesting that themir-192 miRNA is involved in mitosis, and that cells making it through afirst round of cell division may harbor errors that cause them to arrestduring a new round of DNA replication. A uniform 2′-MOE oligomericcompound ISIS Number 338665 targeting the mir-192-1 precursor was alsoobserved to induce a G2/M-phase arrest at both time points.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328744(SEQ ID NO: 653), targeting the mir-182 miRNA, was observed to arrest ordelay the cell cycle in G2/M-phase at 48-hours after oligonucleotidetreatment, suggesting that the mir-182 miRNA plays a role in regulatingmitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328421(SEQ ID NO: 530), targeting miR-30d was also observed to arrest or delaycells mainly in the G2/M-phase at the 24-hour time point and at both theS- and G2/M-phases at the 48-hour time point after oligonucleotidetreatment, suggesting that the mir-30d miRNA is involved in mitosis, andthat a cell division error arising from the first round of division mayallow cells to pass through mitosis and initiate a second round ofdivision, but then a cell cycle checkpoint is set off before the cellsare able to complete DNA synthesis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328403(SEQ ID NO: 512), targeting mir-106 was also observed to arrest or delaycells in the G2/M-phase at the 24-hour time point and at both the S- andG2/M-phases at the 48-hour time point after oligonucleotide treatment,suggesting that the mir-106 miRNA is involved in mitosis, and that acell division error arising from the first round of division may allowcells to pass through mitosis and initiate a second round of division,but then a cell cycle checkpoint is set off before the cells are able tocomplete DNA synthesis. Interestingly, the cell cycle regulatorytranscription factor E2F1 mRNA is reported to be a target of the mir-106miRNA (Lewis et al., Cell, 2003, 115, 787-798).

Treatment of HeLa cells with the oligomeric compound ISIS Number 328759(SEQ ID NO: 668), targeting the mir-216 miRNA, was observed to arrest ordelay the cell cycle in G2/M-phase at both 24- and 48-hours afteroligonucleotide treatment, suggesting that the mir-216 miRNA plays arole in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328342(SEQ ID NO: 451), targeting the mir-203 miRNA, was observed to arrest ordelay the cell cycle in G2/M-phase at both 24- and 48-hours afteroligonucleotide treatment, suggesting that the mir-203 miRNA plays arole in regulating mitosis.

Treatment of HeLa cells with the oligomeric compound ISIS Number 328707(SEQ ID NO: 616), targeting miR-100-1 was also observed to arrest ordelay cells mainly in the G2/M-phase at both 24- and 48-hours afteroligonucleotide treatment, suggesting that the miR-100-1 miRNA plays arole in regulating mitosis.

Dose Responsiveness:

In accordance with the present invention, certain oligomeric compoundstargeting miRNAs were selected for dose response studies. Using the cellcycle assay described above, the cell cycle profiles of HeLa or A549cells treated with varying concentrations of oligomeric compounds of thepresent invention were assessed.

HeLa cells were treated with 25-, 50-, 100- or 150 nM of the oligomericcompounds ISIS

Numbers 327902 (SEQ ID NO: 320) and 328383 (SEQ ID NO: 492), bothtargeted against mir-192, and ISIS 327905 (SEQ ID NO: 323), targetingmir-205, and ISIS 328403 (SEQ ID NO: 512), targeting mir-106. Cellstreated with increasing concentrations of oligomeric compounds werecompared to untreated cells, to assess the dose-dependency of theobserved delay or arrest. The random-mer ISIS 29848 was used as anegative control. Cells were prepared for flow cytometry 48-hours afteroligonucleotide treatment, as described, supra. Oligomeric compoundstargeted to miRNAs were tested in quadruplicate, and ISIS 29848 wastested in triplicate; data is presented as an average of the replicates.Results of these dose response studies are shown in Table 33, where dataare expressed as percentage of cells in each phase.

TABLE 33 Dose response of oligomeric compounds targeting miRNAs on cellcycling (48 hours) Dose oligomeric % cells in cell cycle phase ISIS #compound SubG1 G1 S G2/M Aneuploid Untreated  25 nM 1.3 56 24 20 12control  50 nM 1.4 56 24 20 14 (UTC) 100 nM 1.6 57 24 19 11 150 nM 1.657 23 20 15  29848  25 nM 2.0 55 25 20 12  50 nM 1.5 56 25 19 12 100 nM3.2 52 28 20 13 150 nM 4.2 48 31 21 15 327902  25 nM 1.6 57 23 19 13  50nM 2.4 51 30 20 14 100 nM 3.1 43 30 27 11 150 nM 6.3 36 36 28 12 327905 25 nM 1.7 57 24 18 12  50 nM 2.1 50 30 20 12 100 nM 2.5 46 30 24 14 150nM 4.5 38 38 24 12 328383  25 nM 1.9 57 25 18 12  50 nM 1.3 56 25 18 13100 nM 9.3 36 34 30 10 150 nM 11.8 29 36 34 11 328403  25 nM 1.5 58 2418 13  50 nM 1.1 53 27 20 14 100 nM 3.5 48 29 23 11 150 nM 8.2 37 40 2413

From these data, it was observed that 48-hours after treatment of HeLacells with increasing doses of each of these four oligomeric compoundstargeting miRNAs, a dose-responsive delay or arrest resulted, exhibitedas an increasing percentage of cells in the S- and G2/M-phases of thecell cycle. Concomittent decreases in the percentage of cells inG1-phase of the cell cycle and increases in the percentage ofhypodiploid (subG1) cells were also observed. Likewise, adose-responsive G2/M delay or arrest was observed in A549 cells treatedwith 25-, 50-, 100-, or 150 nM of the oligomeric compounds ISIS 327902,ISIS 328383 and ISIS Number 328342.

In a further study, A549 cells were treated with 25-, 50-, 100- or 150nM of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464)targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targetedagainst mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a,and ISIS 340350 (SEQ ID NO: 855) targeted against mir-95 (Mourelatos).Cells treated with increasing concentrations of oligomeric compoundswere compared to untreated cells, to assess the dose-responsiveness ofthe observed delay or arrest. The random-mer ISIS 29848 was used as anegative control. Cells were prepared for flow cytometry 24-hours afteroligonucleotide treatment. Results of these dose response studies areshown in Table 34, where data are expressed as percentage of cells ineach phase relative to the untreated control cells in that phase.

TABLE 34 Dose response of oligomeric compounds targeting miRNAs on cellcycling (24 hours) Dose oligomeric % cells in cell cycle phase ISIS #compound SubG1 G1 S G2/M Aneuploid 29848  25 nM 90 121 86 87 76  50 nM91 116 88 93 90 100 nM 272 125 74 112 116 150 nM 507 126 71 119 84338637  25 nM 89 100 99 101 99  50 nM 86 110 89 107 120 100 nM 67 126 73115 146 150 nM 216 123 66 144 135 338769  25 nM 62 101 94 114 101  50 nM82 114 81 122 132 100 nM 130 124 75 113 157 150 nM 341 117 71 145 184338836  25 nM 76 97 103 97 99  50 nM 232 113 89 98 111 100 nM 68 117 80116 153 150 nM 178 117 69 149 114 340350  25 nM 91 102 100 95 120  50 nM158 128 67 126 80 100 nM 267 125 60 155 107 150 nM 402 128 40 211 108

From these data, it was observed that 24-hours after treatment of A549cells with increasing doses of the oligomeric compounds ISIS Numbers338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ IDNO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663)targeted against mir-23a, and ISIS 340350 (SEQ ID NO:855) targetedagainst mir-95 (Mourelatos), a dose-responsive delay or arrest resulted,exhibited as an increasing percentage of cells in the G2/M-phases of thecell cycle. Concomittent decreases in the percentage of cells in S-phaseof the cell cycle and increases in the percentage of hypodiploid (subG1)cells were also observed.

In further studies, additional cell lines were treated with oligomericcompounds targeted against miRNAs to assess the effects of eacholigomeric compound on cell cycling. BJ, B16, T47D, and HeLa cells werecultured and transfected as described above. T47D cells are deficient inp53. T47Dp53 cells are T47D cells that have been transfected with andselected for maintenance of a plasmid that expresses a wildtype copy ofthe p53 gene (for example, pCMV-p53; Clontech, Palo Alto, Calif.), usingstandard laboratory procedures. BJ cells were treated with 200 nM ofeach oligomeric compound, and T47D, T47Dp53, HeLa, and B16 cells weretreated with 150 nM of each oligomeric compound. The human foreskinfibroblast BJ cell line represents a non-cancer cell line, while HeLa,T47D, T47Dp53 cells and the mouse melanoma cell line B16-F10 representcancerous cell lines. For comparison, oligomeric compounds ISIS 183891(SEQ ID NO: 847) and ISIS 285717 (TCGGTTCTTTCCAAGGCTGA; hereinincorporated as SEQ ID NO: 857), both targeting the kinesin-like 1/Eg5mRNA, involved in cell cycling, were used as positive controls. Therandom-mer ISIS 29848 was used as a negative control. Additionally, theoligomeric compounds ISIS Number 25690 (ATCCCTTTCTTCCGCATGTG; hereinincorporated as SEQ ID NO: 858) and ISIS Number 25691(GCCAAGGCGTGACATGATAT; herein incorporated as SEQ ID NO: 859), targetedto nucleotides 3051-3070 and 3085-3104, respectively, of the mRNAencoding the Drosha RNase III (GenBank Accession NM_(—)013235.2,incorporated herein as SEQ ID NO: 860) were also tested. ISIS Number25690 and ISIS Number 25691 are 5-10-52′-MOE gapmer compounds, 20nucleotides in length, with phosphorothioate internucleoside linkagesthroughout the oligomeric compound. All cytidine residues are5-methylcytidines. Transfections were performed using the methodsdescribed herein. Cells were prepared for flow cytometry 48-hours afteroligonucleotide treatment. Results of these studies are shown in Table35, where data are expressed as percentage of cells in each phaserelative to the untreated control cells in that phase.

TABLE 35 Effects of oligomeric compounds targeting miRNAs on cellcycling (48 hours) SEQ % cells in cell cycle phase Cell ID Sub line ISIS# NO target G1 G1 S G2/M aneuploid BJ 29848 737 N/A 187 100 99 100 105B16 29848 737 N/A 143 98 98 110 99 HeLa 29848 737 N/A 403 83 113 106 155T47D 29848 737 N/A 86 95 113 98 155 T47Dp53 29848 737 N/A 173 121 75 9793 BJ 183891 847 kinesin-like 1/eg5 422 58 173 287 158 B16 285717 857kinesin-like 1/eg5 627 72 78 220 178 HeLa 183891 847 kinesin-like 1/eg51237 22 95 211 161 T47D 183891 847 kinesin-like 1/eg5 85 55 84 156 161T47Dp53 183891 847 kinesin-like 1/eg5 351 71 53 189 84 HeLa 25690 858Drosha, RNAse III 64 119 89 87 140 T47D 25690 858 Drosha, RNAse III 9797 80 113 140 T47Dp53 25690 858 Drosha, RNAse III 193 97 108 114 144 BJ25691 859 Drosha, RNAse III 183 94 116 125 209 B16 25691 859 Drosha,RNAse III 316 116 83 99 105 HeLa 25691 859 Drosha, RNAse III 881 53 141113 203 T47D 25691 859 Drosha, RNAse III 125 94 104 104 203 T47Dp5325691 859 Drosha, RNAse III 212 130 66 93 95 HeLa 338797 624hypothetical 144 104 89 115 125 miRNA-144 HeLa 338666 493 hypotheticalmiRNA 214 92 98 130 151 039 HeLa 338833 660 hypothetical miRNA 255 87100 136 136 181 HeLa 328707 616 mir-100-1 125 103 87 122 140 BJ 328403512 mir-106 81 102 95 92 114 B16 328403 512 mir-106 112 111 88 99 92HeLa 328403 512 mir-106 89 125 89 80 175 T47D 328403 512 mir-106 49 104112 89 175 T47Dp53 328403 512 mir-106 140 114 87 94 89 HeLa 341787 852MiR-125a, Mouse 324 88 96 145 177 T47D 328687 596 mir-130b 142 101 92115 169 T47Dp53 338769 596 mir-130b 116 103 87 123 87 B16 327933 351mir-145 104 109 84 116 130 BJ 345395 351 mir-145 132 100 97 104 115 B16345395 351 mir-145 147 106 87 115 150 HeLa 345395 351 mir-145 87 108 9695 139 BJ 328744 653 mir-182 125 94 111 127 158 B16 328744 653 mir-182153 108 87 110 115 HeLa 328744 653 mir-182 1057 53 110 213 178 T47D328744 653 mir-182 85 90 87 118 191 T47Dp53 328744 653 mir-182 90 130 59101 100 BJ 327902 320 mir-192-1 91 99 88 108 82 B16 327902 320 mir-192-1151 112 88 98 101 HeLa 327902 320 mir-192-1 94 108 96 93 162 T47D 327902320 mir-192-1 102 75 120 116 162 T47Dp53 327902 320 mir-192-1 155 100 98102 97 HeLa 338665 492 mir-192-1 322 92 92 142 138 HeLa 328342 451mir-203 103 96 89 138 96 BJ 327905 323 mir-205 105 100 77 109 102 B16327905 323 mir-205 142 107 89 106 94 HeLa 327905 323 mir-205 55 108 9990 164 T47D 327905 323 mir-205 81 97 101 103 164 T47Dp53 327905 323mir-205 109 112 80 104 103 HeLa 338678 505 mir-205 129 103 94 105 132 BJ328759 668 mir-216 164 91 117 141 160 B16 328759 668 mir-216 132 104 91110 126 HeLa 328759 668 mir-216 797 40 82 203 223 T47D 328759 668mir-216 123 86 87 122 223 T47Dp53 328759 668 mir-216 423 99 93 108 109HeLa 327896 314 mir-22 95 103 94 106 144 HeLa 338836 660 mir-23a 303 9796 121 114 HeLa 328084 743 mir-30a 286 89 92 153 125 HeLa 340350 855mir-95 132 101 94 112 177 (Mourelatos)

When treatment of cells with oligomeric compounds resulted in greaterthan 750% cells in subG1 phase, these oligomeric compounds were deemedto be “hits,” in that they appear to cause an increase in apoptosis,resulting in hypodiploid DNA contents. When treatment of cells witholigomeric compounds resulted in greater than 140% cells in G1-phase,these oligomeric compounds were deemed “hits,” as they appeared to causean arrest or delay in G1-phase and/or blocked entry into S-phase of thecell cycle. When treatment of cells with oligomeric compounds resultedin greater than 140% cells in S-phase, these oligomeric compounds weredeemed “hits,” as they appeared to cause an arrest or delay in DNAsynthesis. When treatment of cells with oligomeric compounds resulted ingreater than 140% cells in G2/M phase, these oligomeric compounds weredeemed “hits,” as they appeared to cause an arrest or delay in thetransition into mitosis, and/or in cell division, itself.

From these data, it was observed that 48-hours after treatment of thevarious cell lines with the oligomeric compounds, ISIS Number 183891targeting the kinesin-like 1/Eg5 mRNA results in a delay or arrest inG2/M phase of the cell cycle for all cell lines. Treatment of HeLa cellswith ISIS Number 25691, targeted against the Drosha RNase III mRNA,resulted in an increased percentage of cells in S-phase as well as asignificant percentage of cells in the subG1 and aneuploid categories,indicating that this oligomeric compound may interfere with DNAreplication and/or maintenance of the integrity of the proper complementof genetic material.

In HeLa cells, ISIS 341787 (SEQ ID NO: 852) targeted against mir-125a(mouse), resulted in an arrest or delay in G2/M as well as an increasedpercentage of cells in the subG1 and aneuploid categories, indicatingthat this oligomeric compound may interfere with cell division and equalchromosome segregation during mitosis.

In HeLa cells treated with ISIS 328744 (SEQ ID NO: 653) targeted againstmir-182, an increase in the percentage of cells in the G2/M-phase of thecell cycle as well as in the subG1 category was observed, indicatingthat this oligomeric compound may interfere with cell division and equalchromosome segregation during mitosis. Notably, genetically normal cells(BJ and T47Dp53cells) were not affected by ISIS Number 328744,indicating that the oligomeric compound targeting miR-182 mayselectively cause a cell cycle delay or arrest in cancer cells and notnormal cells, and suggesting that this compound may be particularlyuseful as a therapeutic agent in the treatment of hyperproliferativedisorders such as cancer.

In HeLa cells treated with ISIS 328759 (SEQ ID NO: 668) targeted againstmir-216, a delay or arrest resulted in the G2/M-phase of the cell cyclewas observed, as well as an increase in the percentage of cells in thesubG1 and aneuploid categories, indicating that this oligomeric compoundmay interfere with cell division and equal chromosome segregation duringmitosis.

Thus, it was observed that treatment of HeLa cells with oligomericcompounds targeting miRNAs is a effective means of identifying compoundsthat can block progression through various stages of the cell cycle.Notably, a transient increase in G1-phase was observed 24 hours aftertreatment of HeLa cells with oligomeric compounds targeting miRNAs; forexample, oligomeric compounds ISIS Numbers 338769, 338836, 340350, and338637 caused a transient increase in the percentage of cells delayed orarrested in G1-phase at the 24-hour time point, which, by the 48-hourtime point, had shifted to a delay or arrest in S-phase. It was alsonoted that multiple oligomeric compounds targeting the same miRNA havethe same effect on cell cycling. It was also observed that uniform2′-MOE as well as 2′-MOE chimeric gapmer oligomeric compounds targetingthe mature miRNA, as well as uniform 2′-MOE oligomeric compoundstargeting the pri-miRNA often have the same effect.

Oligomeric compounds that delay, arrest or prevent cell cycleprogression or induce apoptosis may be useful as therapeutic agents forthe treatment of hyperproliferative disorders, such as cancer, cancer,as well as diseases associated with a hyperactivated immune response.

It is understood that BJ, B16, HeLa, A549, HMECs, T47D, T47Dp53, MCF7 orother cell lines can be treated with oligomeric compounds designed tomimic miRNAs in studies to examine their effects on progression throughthe cell cycle. Such oligomeric compounds are within the scope of thepresent invention.

Example 25 A Bioinformatic Approach to Identification of miRNA Targets

Several candidate RNA transcripts identified using the RACE-PCR methodsdescribed in Example 20 were the basis for a bioinformatic analysis ofpredicted targets bound to and/or potentially regulated by miRNAs. Thecomplementarity between the miRNA used as a primer and the 3′-UTR of theRNA transcript identified by RACE-PCR was assessed using severalmethods. Transcripts identified by RACE-PCR were also analyzed using theFASTA sequence alignment program (accessible through the internet at,for example, www.ebi.ac.uk/fasta33) to find the best alignment betweencomplementary sequences of the transcript and the miRNA used as a primerfor RACE-PCR. When, using the default parameters, the FASTA alignmentprogram resulted in the identification of the actual primer binding site(PBS) within the 3′-UTR of the RNA transcript as the sequence mostcomplementary to the miRNA used as a primer in the RACE-PCR method, thecandidate miRNA target transcript was specified by a plus sign (forexample, see the “mir-143/PBS complementary?” column in Table 36 below).When the FASTA program failed to align the actual PBS with the sequencemost complementary to the miRNA used in the RACE-PCR, the candidatemiRNA target transcript was specified by a minus sign. When the FASTAprogram could be made to align with the sequence most complementary tothe miRNA used in the RACE-PCR by decreasing the stringency of the FASTAprogram parameters, the candidate miRNA target transcript was specifiedby “±”.

A global alignment was also performed to assess whether the sequence ofthe PBS within the RNA transcript identified by RACE-PCR was conservedbetween human and mouse orthologs of the RNA target. For example, inTable 36, below, strong conservation of PBS in the human and murineorthologs (homology from 80-100%) was indicated by a plus sign; moderateconservation (homology between 70-80%) was indicated by “±”, and a minussign indicates homology below 70%.

A variety of algorithms can be used to predict RNA secondary structuresbased on thermodynamic parameters and energy calculations. For example,secondary structure prediction can be performed using either M-fold orRNA Structure 2.52. M-fold can be accessed through the Internet at, forexample, www.ibc.wustl.edu/-zuker/ma/form2.cgi or can be downloaded forlocal use on UNIX platforms. M-fold is also available as a part of GCGpackage. RNA Structure 2.52 is a windows adaptation of the M-foldalgorithm and can be accessed through the Internet at, for example,128.151.176.70/RNAstructure.html. The RNA Structure 2.52 program wasused to analyze a series of 30-base fragments spanning the entire lengthof the human RNA transcript and their potential to hybridize with themiRNA used as a primer in the RACE-PCR, allowing the prediction of thelowest absolute free energy peak representing the most likely site ofhybridization (including bulged regions) between the miRNA and the RNAtarget. If the free energy peak representing the hybridization betweenthe miRNA and the PBS of the RNA transcript identified by RACE-PCR wasamong the top five peaks predicted by the RNA Structure 2.52 program,the transcript was given a plus sign, “+”. If the free energy peakrepresenting the hybridization between the miRNA and the PBS was in thetop five to ten peaks predicted by RNA Structure 2.52, the transcriptwas given a plus/minus sign, “−”, and if the peak representing thehybridization between the miRNA and the PBS was below the top ten peakspredicted by RNA Structure 2.52, the transcript was given a minus sign,“−”.

A list of the RNA transcript targets identified by RACE-PCR employingthe mir-143 miRNA as a specific primer is shown in Table 36.

TABLE 36 Potential RNA targets of the mir-143 miRNA RNA SEQ ID Structuremir-143/PBS RNA transcript target NO: PBS conserved? peak?complementary? Matrix 819 + − + metalloproteinase 2 Sec24 829 − +/− +Tripartite motif- 828 +/− + +/− containing 32 RAN 824 +/− + + Cystatin B802 − + + Glucocorticoid 839 + +/− + induced transcript 1 Proteinphosphatase 2 809 + + + Polycystic kidney 822 − − − disease 2Mannose-6-phosphate 801 +/− − + receptor Mitotic control 817 + + −protein dis3 homolog Chromosome 14 ORF 103 813 + +/− − Rho GDPdissociation 823 − − − inhibitor beta Glyoxalase I 816 + + + Zinc fingerprotein 818 + +/− + 36, C3H type-like 1 LIM domain only 4 804 + + +

Note that four genes (Sec24, cystatin B, polycystic kidney disease 2,and Rho GDP dissociation inhibitor beta) did not have murine orthologsto compare in a global analysis of the PBS. Because these RNAtranscripts were identified as being bound by the mir-143 miRNA used asa primer in the RACE-PCR approach previously described, the mir-143miRNA is predicted to serve a regulatory role in expression or activityof one or more or all of these RNA transcripts. Of particular note arethree targets, protein phosphatase 2, glyoxalase I, and LIM domain only4 (LMO4) mRNAs, for which all three analyses yielded a positive result.That all three parameters assessed yielded a positive result suggeststhat these mRNAs are probable targets of mir-143.

The well-studied C. elegans lin-4 miRNA interaction with its lin-28 mRNAtarget was used as the starting point for a bioinformatics approach tothe identification of miRNA binding sites in target nucleic acids. Lin-4has been experimentally determined to bind at a single site on thelin-28 mRNA. Herein, as a primary determinant of miRNA-targetinteractions, it was hypothesized that the bimolecular hybridizationfree energies (ΔG°₃₇) of the interaction of the miRNA with a true targetsite would be more negative than the ΔG°₃₇ of other interactions of themiRNA with other binding sites. The nucleotide sequence of the lin-28mRNA was assessed by computationally deriving 30-nucleotide windows,starting with the first nucleotide of the sequence and defining thefirst nucleotide in each window by shifting 1 nucleotide in the 3′direction. Each window was assessed by hybridizing the 30-nucleotidesequence in the window with the lin-4 miRNA and disallowing unimolecularinteractions, thereby spanning the entire length of the lin-28 mRNA, andthe resulting ΔG°₃₇ value was plotted against the start position of thewindow. It was observed that the bimolecular hybridization between thetrue lin-4 binding site and the lin-28 mRNA had the lowest ΔG°₃₇ value,supporting our hypothesis and our bioinformatic approach to theidentification of miRNA binding sites in target nucleic acids.

The mitogen-activated protein kinase 7/extracellular signal-regulatedkinase 5 (ERK5) (GenBank Accession NM_(—)139032.1, incorporated hereinas SEQ ID NO: 861) mRNA transcript was previously computationallypredicted to be regulated by mir-143 miRNA binding in the 3′-UTR regions(Lewis et al., Cell, 2003, 115, 787-798). In order to identify mir-143binding sites in the ERK5 mRNA, a bimolecular hybridization free energyassessment was performed by performing a hybridization walk to assesspossible mir-143 binding sites along the entire length of the ERK5 mRNA.A strong negative ΔG°₃₇ value (−20.1) was found at the previouslypredicted mir-143 binding site in the 3′-UTR, lending further support toour method. Surprisingly, two additional, and novel, mir-143 bindingsites with more negative ΔG°₃₇ values, as well as a third mir-143binding site with a comparable ΔG°₃₇ value were also identified. Usingthe ERK5 sequence (GenBank Accession NM_(—)139032.1) as a reference,these binding sites encompass nucleotides 937-966 with a ΔG°₃₇ value of(−22.8), nucleotides 2041-2070 with a ΔG°₃₇ value of (−20.6) andnucleotides 2163-2192 with a ΔG°₃₇ value of (−19.3). See FIG. 1. Thus,three novel mir-143 binding sites (and, thus, a potential regulatorysites) were identified within the coding sequence of the ERK5 gene.Thus, this method of screening for miRNA binding sites by a bimolecularhybridization free energy assessment can be used to confirm previouslypredicted sites, and further allows the identification of novel miRNAtarget nucleic acid binding sites. It is believed that this method maymore closely mimic the energetic mechanism by which a miRNA scans atarget nucleic acid to find its interaction site. In subsequentexperiments, the predicted mir-143 binding sites within the ERK5 codingsequence were also tested using the reporter system described below.

Example 26 Northern Analysis of miRNA Expression

As described in the adipocyte differentiation assay, the oligomericcompounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b, andISIS Number 327876 (SEQ ID NO: 294), targeted to mir-29b-1, were foundto reduce the expression of several hallmark genes of adipocytedifferentiation, indicating that mir-23b and mir-29b-1 may play a rolein adipocyte differentiation, and that oligomeric compounds targetingthese miRNAs may be useful as agents blocking cellular differentiation.Therefore, the expression of mir-23b and mir-29b was assessed byNorthern blot of total RNA from multiple tissues. To detect the mir-23band mir-29b-1 targets, target specific DNA oligonucleotide probes withthe sequences GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) andAACACTGATTTCAAA TGGTGCTA (SEQ ID NO: 294), respectively, weresynthesized by IDT (Coralville, Iowa). The oligo probes were 5′end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP (Promega). Tonormalize for variations in loading and transfer efficiency membranesare stripped and probed for U6 RNA. Total RNA from mouse and humantissues as well as total RNA from human adipocytes and HepG2 cells wasprobed in Northern blot analyses, using methods described in Example 14.

By Northern analyses, the mir-23b miRNA was found to be most highlyexpressed in human kidney tissue as well as in adipose tissue from ob/obmice, and was also highly expressed in human liver, adipocytes,preadipocytes and HepG2 cells. Moderate expression of mir-23b was alsonoted in murine kidney tissue. The mir-29b-1 miRNA was found to be mosthighly expressed in human and mouse kidney, and was also expressed inhuman liver, adipocytes, preadipocytes, and HepG2 cells, as well as inmurine adipose tissue and liver. Levels of both the mir-23b andmir-29b-1 miRNAs were also found to be upregulated in humandifferentiated adipocytes.

Similarly, target specific DNA oligonucleotide probes for mir-16,mir-15a, and let-7a were designed and used in Northern blot analyses toassess expression of these miRNAs in human and mouse tissues. The mir-16and mir-15a miRNAs were each found to be most highly expressed in humanspleen, heart, testes, and kidney, and expression was also observed inliver as well as HEK293 and T47D cells. Additionally, low levels ofexpression of the mir-16 miRNA were observed in NT2 cells. The let-7amiRNA was most highly expressed in human and murine kidney, andexpression was also observed in human and murine liver. Additionally,low levels of let-7a expression were found in HepG2 cells.

To detect the mir-21 miRNA in Northern blot analyses, a target specificDNA oligonucleotide probe with the sequences TCAACATCAGTCTGATAAGCTA (SEQID NO: 335) was synthesized by IDT (Coralville, Iowa). The oligo probeswere 5′ end-labeled with T4 polynucleotide kinase with (γ-³²P) ATP(Promega). Twenty micrograms of total RNA from human PromyelocyticLeukemia HL-60 cells, A549, HeLa, HEK293, T47D, HepG2, T-24, MCF7, andJurkat cells was fractionated by electrophoresis through 15% acrylamideurea gels using a TBE buffer system (Invitrogen). RNA was transferredfrom the gel to HYBOND™-N+nylon membranes (Amersham Pharmacia Biotech,Piscataway, N.J.) by electroblotting in an Xcell SureLock™ Minicell(Invitrogen). Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.)and then probed using Rapid Hyb buffer solution (Amersham) usingmanufacturer's recommendations for oligonucleotide probes. To normalizefor variations in loading and transfer efficiency membranes are strippedand probed for U6 RNA. High levels of expression of mir-21 were observedin A549 and HeLa cells; in fact, levels of mir-21 expression were notedto be among the highest of any of the miRNAs observed in HeLa cells.

Example 27 Reporter Systems for Assaying Activity of OligomericCompounds Targeting or Mimicking miRNAs

Reporter systems have been developed herein to assess the ability ofmiRNA mimics to provoke a gene silencing response and to assess whetherantisense oligomeric compounds targeting miRNAs can inhibit genesilencing activity. The T-REx™-HeLa cell line (Invitrogen Corp.,Carlsbad, Calif.) was used for either stable or transient transfectionswith plasmids constitutively expressing miRNAs, pre-miRNAs, pri-miRNAsor mimics thereof, and, in some cases, antisense oligomeric compoundstargeting the expressed miRNA were also transfected into the cells. Itis understood that other mammalian cells lines can also be used in thisreporter system. T-REx™-HeLa cells were routinely cultured in DMEM, highglucose (Invitrogen Corporation, Carlsbad, Calif.), supplemented with10% fetal bovine serum (Invitrogen Corporation). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were harvested when they reached 90% confluence, andon the day before transfection with expression or reporter plasmids(described in detail below), the T-REx™-HeLa cells were seeded onto24-well plates at 50,000 cells/well. The following day, cells weretransfected according to standard published procedures with variouscombinations of plasmids using 2 μg Lipofectamine™ 2000 Reagent(Invitrogen) per μg of plasmid DNA. When transfecting oligomericcompounds, 1-3 μg of Lipofectamine™ 2000 Reagent was used per 100 nMoligomeric compound.

Plasmids used are as follows: The pcDNA3.1©/NT-GFP (Invitrogen) plasmid,containing a CMV promoter controlling expression of a GFP reportersequence at the N-terminus of the transcription start site was used as acontrol plasmid. The pcDNA3.1©/NT-GFP-mir-143 sensor plasmid contains(in addition to the elements above) three 22-nucleotide sites encodingthe mir-143 miRNA binding site, downstream of the GFP coding sequenceand upstream of the polyadenylation signal. The pCR3-pri-mir-143 plasmid(“pri-mir-143”) is a CMV promoter-driven constitutive expression plasmidwhich expresses the 110-nucleotide mir-143 pri-miRNA sequence (SEQ IDNO: 38), to act as a mir-143 pri-miRNA mimic. The pCR3-pri-mir control(“pri-mir-control”) is a CMV promotor-driven constitutive expressionplasmid which is designed to express a similar 110-nucleotide pri-miRNAsequence (AGCAGCGCAGCGCCCTGTCTCCCAGCCAAGGTGGAACCTTCTGGGAAGCGGTCAGTTGGGAGTCCCTTCCCTGAAGGTTCCTCCTTGGAAGAGAGAAGTTGTTCTG CAGC; SEQID NO: 862) wherein the mature mir-143 sequence has been replaced withan unrelated sequence and the predicted complementary strand opposite itwithin the pri-miRNA structure is replaced with a nearly complementarysequence in order to preserve the stem loop as well as the bulgestructure of the natural mir-143 pri-miRNA. Additionally, in order totest the effect of an oligomeric compound targeting a miRNA, theT-REx™-HeLa cells were also transfected with the uniform 2′-MOEphosphorothioate (PS) antisense oligomeric compound ISIS Number 327901(SEQ ID NO: 319), targeted to mir-143 previously described.

Twenty-four hours post-transfection, cells were trypsinized and GFPfluorescence was analyzed by flow cytometry. Results are shown in Table37.

TABLE 37 Mean GFP fluorescence after transfection of T-REx ™-HeLa cellsTreatment pri-mir GFP GFP mir-143 327901 Mean control pri-mir-143control sensor oligo fluorescence − − − − − 2.2 + − − − − 2.7 − + − − −2.6 − − + − − 7.9 + − + − − 22.7 − + + − − 9.6 − − − + − 12.4 + − − + −21.8 − + − + − 5.3 − + − + + 4.1 − − − + + 4.2 − + − − + 3.7

Plus signs, “+”, indicate the presence of the expression plasmid oroligomeric construct in transfectants; minus signs “−”, indicate theabsence of same. Mean fluorescence is measured in arbitrary units.

In cells transfected with the sensor plasmid and expressing the mir-143pri-miRNA mimic from the pCR3-pri-mir-143 plasmid, the mir-143 miRNA isexpected to be processed endogenously, allowing it to bind as a maturemiRNA to the RNA transcript encoding GFP and containing the mir-143binding sites expressed from the reporter plasmid, resulting in cleavageof the reporter transcript and a decrease in fluorescence as compared tothe control plasmid. From the data shown in Table 37, it was observedthat expression of the pCR3-pri-mir-143 plasmid results in an inhibitionof expression of GFP indicated by a decrease in fluorescence produced bythe pcDNA3.1/NT-GFP-mir-143 sensor plasmid, whereas expression of thepCR3-pri-mir control plasmid had no effect on GFP reporter expression.Neither the pCR3-pri-mir control nor the pCR3-pri-mir-143 plasmid hadany inhibitory effect on GFP expression from the pcDNA3.1©/NT-GFPcontrol plasmid. Thus, the mir-143 pri-miRNA mimic oligomeric compoundsilences the expression of RNA transcribed from a reporter plasmidcontaining mir-143 target sites.

In a further study, T-REx™-HeLa cells transfected with thepcDNA3.1/NT-GFP-mir-143 sensor plasmid were treated at various dosageswith the following oligomeric compounds: 1) a double-stranded RNAoligomeric compound (“ds-mir-143”) composed of ISIS Number 342199(TGAGATGAAGCACTGTAGCTCA; SEQ ID NO: 220) representing the mir-143 sensesequence, hybridized to its perfect complement ISIS Number 342200(TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319); 2) a negative control dsRNA(“ds-Control”), representing a 10-base mismatched sequence antisense tothe unrelated PTP1B mRNA, composed of ISIS Number 342427(CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 863) hybridized to its perfectcomplement ISIS Number 342430 (GGAGGAACCTTCAGGGAAGG; SEQ ID NO: 864); 3)the pCR3-pri-mir-143 expression plasmid (“pCR3-pri-mir-143”) whichexpresses the 110-nucleotide mir-143 pri-miRNA; 4) the pCR3-pri-mircontrol (“pri-mir-control”); 5) an in vitro transcribed RNA oligomericcompound (“hairpin mir-143”) representing the 10 bp fragment of themir-143 pri-miRNA molecule (SEQ ID NO: 38) plus an additional twocytosine nucleobases from the T7 promoter at the 5′ end; and 6) an invitro transcribed RNA oligomeric compound (“hairpin control”) (SEQ IDNO: 862) representing a similar hairpin structure except that the maturemir-143 sequence and its complementary sequence within the pri-miRNAhairpin structure were replaced with sequences unrelated to mir-143. TheRNA hairpin oligomeric compounds were in vitro transcribed using theMAXIscript Kit (Ambion Inc., Austin, Tex.) according to themanufacturer's protocol, beginning with a DNA template amplified by PCR.GFP fluorescence of treated cells was assessed using the methodsdescribed above, and it was observed that the ds-mir-143 oligomericcompound mimic inhibited expression of GFP from the sensor plasmid in adose dependent manner. In a further embodiment, pcDNA3.1©/NT-GFP-mir-143sensor-expressing cells treated with 20 nM mir-143 dsRNA oligomericcompound were additionally treated with 4-, 20- or 100 nM uniform 2′-MOEoligomeric compound ISIS Number 327901 (SEQ ID NO: 319), or 4-, 20- or100 nM uniform 2′-MOE scrambled mir-143 control ISIS Number 342673 (SEQID NO: 758) to assess the ability of compounds to inhibit the inhibitoryeffect of the mir-143 dsRNA mimic. At all three concentrations, theoligomeric compound ISIS Number 327901 targeting mir-143 blocked theinhibitory effect of the mir-143 dsRNA oligomeric compound, exhibited asa recovery of GFP fluorescence.

In one embodiment, an expression system based on the pGL3-Control(Promega Corp., Madison Wis.) vector containing a CMV promotercontrolling expression of a luciferase reporter sequence was used intransient transfections of HeLa cells with plasmids expressing miRNA orpri-miRNA mimics. To assess the ability of miRNA mimics to bind andregulate the expression of the luciferase reporter gene, two reporterplasmids were constructed: 1) a synthetic DNA fragment comprising twosites perfectly complementary to mir-143 were inserted into thepGL3-Control luciferase reporter vector, to create the pGL3-mir-143sensor plasmid, and 2) a DNA fragment comprising the 3′-UTR of the LIMdomain only 4 (LMO4) gene (predicted to be regulated by mir-143) wasinserted into pGL3-Control to create pGL3-LMO4; this fragment wasPCR-amplified using a primer beginning at position 1261 of the LMO4sequence (GenBank Accession NM_(—)006769.2, incorporated herein as SEQID: 809) and the downstream primer hybridizing to the poly-A tail. Ineach of these plasmids, the target site was placed downstream of theluciferase coding sequence and upstream of the polyadenylation signal inthe 3′-UTR of the luciferase reporter vector. The unmodifiedpGL3-Control luciferase reporter vector was used as a control.

HeLa cells were routinely cultured and passaged as described, and on theday before transfection with expression or reporter plasmids, the HeLacells were seeded onto 24-well plates 50,000 cells/well. Cells weretransfected according to standard published procedures with variouscombinations of plasmids using 2 μg Lipofectamine™ 2000 Reagent(Invitrogen) per μg of plasmid DNA, or, when transfecting oligomericcompounds, 1.25 μg of Lipofectamine™ 2000 Reagent per 100 nMoligonucleotide or double-stranded RNA. The luciferase signal in eachwell was normalized to the Renilla luciferase (RL) activity producedfrom a co-transfected plasmid, pRL-CMV, which was transfected at 0.5 μgper well. Cells were treated at various dosages (4 nM, 20 nM, and 100nM) with the following oligomeric compound mimics: 1) “ds-mir-143,” 2)“ds-Control,” 3) “pCR3-pri-mir-143,” or 4) “pri-mir-control,” asdescribed supra. In accordance with methods described in Example 12,supra, a luciferase assay was performed 48-hours after transfection.Briefly, cells were lysed in passive lysis buffer (PLB; Promega), and 20ul of the lysate was then assayed for RL activity using a DualLuciferase Assay kit (Promega) according to the manufacturer's protocol.The results below are an average of three trials and are presented aspercent pGL3-Control luciferase expression normalized to pRL-CMVexpression (RL). The data are shown in Table 38.

TABLE 38 Luciferase assays showing effects of oilgomeric compoundsmimicking mir-143 luciferase expression (% lucif. only control) pGL3-pGL3-mir-143 treatment Control sensor pGL3-LMO4 no luciferase (pRL) 0.30.3 0.4 luciferase (pRL) only 100.0 101.0 100.0 ds-mir-143 (4 nM) 101.514.5 151.6 ds-mir-143 (20 nM) 123.8 8.0 140.1 ds-mir-143 (100 nM) 131.87.1 128.4 ds-Control (4 nM) 133.6 144.5 172.4 ds-Control (20 nM) 126.1169.8 151.6 ds-Control (100 nM) 123.0 151.3 151.5 pCR3-pri-mir-143 (0.25ug) 75.6 58.6 101.9 pCR3-pri-mir-143 76.6 50.7 95.7 precursor (0.5 ug)pCR3-pri-mir-143 (1 ug) 64.7 35.0 82.5 pri-mir control (0.25 ug) 90.378.3 114.8 pri-mir control (0.5 ug) 57.3 61.8 95.4 pri-mir control (1ug) 67.9 64.9 74.8

From these data, it was observed that the mir-143 dsRNA oligomericcompound and the mir-143 pri-miRNA mimic expressed from thepCR3-pri-mir-143 expression plasmid both inhibited luciferase activityfrom the pGL3-mir-143 sensor plasmid in a dose-dependent manner.

In another embodiment, HeLa cells were transfected with 0.03 μgpGL3-mir-143 sensor plasmid and 0.01 μg pRL-CMV plasmid, and, inaddition, (except those samples described below as “without mir-143pri-miRNA,”) were also transfected with 0.01 μg of an expression plasmiddesigned to express a mir-143 pri-miRNA mimic comprising a larger 430-ntfragment of the mir-143 primary miRNA transcript, referred to as“pCR3-pri-mir-143 (430)”(AGGTTTGGTCCTGGGTGCTCAAATGGCAGGCCACAGACAGGAAACACAGTTGTGAGGAATTACAACAGCCTCCCGGCCAGAGCTGGAGAGGTGGAGCCCAGGTCCCCTCTAACACCCCTTCTCCTGGCCAGGTTGGAGTCCCGCCACAGGCCACCAGAGCGGAGCAGCGCAGCGCCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGGTCAGTTGGGAGTCTGAGATGAAGCACTGTAGCTCAGGAAGAGAGAAGTTGTTCTGCAGCCATCAGCCTGGAAGTGGTAAGTGCTGGGGGGTTGTGGGGGGCCATAACAGGAAGGACAGAGTGTTTCCAGACTCCATACTATCAGCCACTTGTGATGCTGGGGAAGTTCCTCTACACAAGTTCCCCTGGTGCCACGATCTGCTTCACGAGTCTGGGCA; SEQ ID NO: 871). It was observed that themir-143 pri-miRNA mimic expressed by pCR3-pri-mir-143 (430) inhibitsluciferase expression from the pGL3-mir-143 sensor plasmid. To furtherevaluate the ability of the mir-143 pri-miRNA mimic to inhibitluciferase activity from the sensor plasmid, and to assess the abilityof oligomeric compounds to interfere with the inhibition of pGL3-mir-143sensor luciferase expression by the mir-143 pri-miRNA mimic,pGL3-mir-143 sensor-expressing HeLa cells treated with pCR3-pri-mir-143(430) were additionally treated with varying concentrations (0-, 6.7- or20 nM) of the following oligomeric compounds: 1) ISIS Number 327901 (SEQID NO: 319), a uniform 2′-MOE oligomeric compound targeting mir-143; 2)ISIS Number 342673 (SEQ ID NO: 758), a uniform 2′-MOE scrambled control;or 3) ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA(mir-129-2). ISIS Numbers 342673 and 327924 were used as negativecontrols. HeLa cells transfected with the pRL-CMV and pGL3-mir-143sensor plasmids, but not treated with the pCR3-pri-mir-143 (430) hairpinprecursor served as a control. In this experiment, the luciferase assaywas performed 24-hours after transfection. The data are presented inTable 39 as relative luciferase activity (normalized to RL expressionlevels). Where present, “N.D.” indicates “no data.”

TABLE 39 Effects of oligomeric compounds on mir-143 pri-miRNAmimic-mediated inhibition of luciferase expression Relative luciferaseactivity SEQ ID Dose of oligomeric compound Treatment NO 0 nM 6.7 nM 20nM 327901 319 0.97 4.0 6.4 342673 758 0.97 1.3 1.5 negative control327924 342 0.97 0.8 1.2 negative control without pCR3-pri-mir-143 (430)N/A 13.8 N.D. N.D.

From these data, it was observed that the oligomeric compound ISISNumber 327901 targeting mir-143 blocked the inhibitory effect of themir-143 pri-miRNA mimic, exhibited as a 4- to 6.4-fold recovery ofluciferase activity in HeLa cells expressing the pGL3-mir-143 sensorplasmid.

More than four-hundred target genes have been predicted to be regulatedby miRNA binding to the 3′-UTR regions of the mRNA transcript (Lewis etal., Cell, 2003, 115, 787-798). For example, at least six genes havebeen reported to bear regulatory sequences in their 3′-UTRs which arepredicted to be bound by the mir-143 miRNA; these include the inwardlyrectifying potassium channel Kir2.2 (GenBank Accession AB074970,incorporated herein as SEQ ID NO: 872), synaptotagmin III (GenBankAccession BC028379, incorporated herein as SEQ ID NO: 873),mitogen-activated protein kinase 7/extracellular signal-regulated kinase5 (ERK5) (GenBank Accession NM_(—)139032.1, SEQ ID NO: 861), proteinphosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB)(GenBank Accession NM_(—)004156.1, SEQ ID NO: 814), glyoxalase I (GLO1)(GenBank Accession NM_(—)006708.1, SEQ ID NO: 821), and LIM domain only4 (LM04) (GenBank Accession NM_(—)006769.2, SEQ ID NO: 809). It shouldbe noted that one third of miRNA targets predicted in the study byLewis, et al. are expected to be false positives (Lewis et al., Cell,2003, 115, 787-798).

Because the present inventors independently identified the PPP2CB andGLO1 genes as potential targets of mir-143 by the RACE-PCR methods asdescribed in Example 20, these targets were selected for further study.In addition, and described in Example 25, a novel mir-143 binding site(and, thus, a potential regulatory site) was identified within thecoding sequence of the ERK5 gene; this predicted mir-143 binding sitewithin the ERK5 coding sequence was also tested in these reportersystems.

In some embodiments, an expression system based on the pGL3-Control(Promega Corp., Madison Wis.) reporter vector and comprising predictedmiRNA binding sites was used in stable transfections of HeLa cells,selecting for cells that have integrated the reporter plasmid into theirgenome. Because pGL3-based reporter vectors have no selectable markerfor antibiotic resistance, a neomycin-resistance (Genetecin) gene wascloned into the pCR2 plasmid (Invitrogen Life Technologies, Carlsbad,Calif.) to create the pCR2-neo plasmid, and pCR2-neo was co-transfectedinto HeLa cells with the pGL3-mir-143-sensor plasmid at a ratio of onepCR2-neo plasmid to ten pGL3-mir-143-sensor plasmids. Co-transfectedcells were then selected for the presence of the Genetecin marker andassayed for luciferase activity; Genetecin-resistant cells are verylikely to have also integrated the luciferase reporter into theirgenome.

Establishment of Stably-Transfected Cells:

One day prior to transfection, approximately 750,000 HeLa cells areseeded onto a 10 cm dish or T-75 flask and grown in complete mediumovernight at 37° C. The next day, 10 μg of pGL3-mir-143-sensor plasmidand 1 μg pCR2-neo are mixed in 2 ml OPTI-MEM™ (Invitrogen Corporation,Carlsbad, Calif.). (Linearization of circular plasmids by digestion withrestriction enzyme may increase the number of stable transfectants perμg transforming DNA, but is not an essential step). 10 μl LIPOFECTIN™reagent (Invitrogen Corporation, Carlsbad, Calif.) is mixed with 2 mlOPTI-MEM™. The plasmid/OPTI-MEM™ and OPTI-MEM™/LIPOFECTIN™ mixtures arethen mixed together, and an additional 11 ml OPTI-MEM™ is added, and theresulting 15 ml cocktail is added to the cells. Cells are incubated inthe plasmid/OPTI-MEM™/LIPOFECTIN™ cocktail for approximately 4 hours at37° C., after which the cocktail is removed and replaced with freshcomplete medium. The following day, cells are trypsinized andtransferred to a T-175 flask. Media containing the selection agent, 500μg/ml G418 (Geneticin; GIBCO/Life Technologies, Gaithersburg, Md.), isadded and cells are grown at 37° C. Cells are re-fed daily with freshmedia containing the selection agent until the majority of the cellsappear to have died off and isolated colonies of neomycin-resistantcells appear. In cases where subcloning is desired, selectedneomycin-resistant cells are trypsinized and plated at a concentrationof 0.5 cells/well in 96-well plates, maintaining the cells in 500 μg/mlG418 selection media.

In one embodiment, five stably-transfected, neomycin-resistant,luciferase-positive, pGL3-mir-143-sensor cell clones were isolated,subcloned and selected for further testing with oligomeric compounds ofthe present invention. Cells stably expressing the luciferase reporterand comprising one or more miRNA binding sites were then transfectedwith oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs inorder to assess the ability of these miRNA mimics to bind and regulatethe expression of the luciferase reporter.

An expression system based on the pGL3-Control (Promega Corp., MadisonWis.) reporter vector and comprising predicted miRNA binding sites wasused in transient transfections of HeLa cells with plasmids expressingoligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in orderto assess the ability of these miRNA mimics to bind and regulate theexpression of the luciferase reporter. The effect of increasing the copynumber of the miRNA-binding site in the target was also tested byincluding multiple binding sites in artificial reporter constructs. Itis understood that the presence of multiple miRNA-binding sites in atarget can include binding sites for different miRNAs.

The following reporter plasmids were constructed by cloning thespecified fragment into the XbaI site of the pGL3-control plasmid,placing the potential miRNA-binding site in the 3′-UTR of the luciferasereporter: The reporter plasmid pGL3-bulge(×3) contains three contiguouscopies of the sequence (TGAGCTACAGCTTCATCTCA; herein incorporated as SEQID NO: 874) which represents a sequence complementary to the mir-143miRNA except that it is missing 2 nucleotides such that the mir-143miRNA is presumed to adopt a bulged structure when it hybridizes to thistarget sequence. The pGL3-GLO1 reporter plasmid contains a DNA fragmentcomprising the 3′-UTR of the GLO1 sequence; this fragment wasPCR-amplified using a primer beginning at nucleotide number 621 of theGLO1 sequence (GenBank Accession NM_(—)006708.1, SEQ ID NO: 821) and thedownstream primer hybridizing to the poly A tail. The pGL3-PP2A reporterplasmid contains a DNA fragment comprising the 3′-UTR of the PP2A gene;this fragment was PCR-amplified using a primer beginning at nucleotidenumber 921 of the PP2A sequence (GenBank Accession NM_(—)004156.1) andthe downstream primer hybridizing to the poly A tail. The reporterplasmid pGL3-ERK5-3′-UTR(×1) contains one copy of the sequenceTATTCTGCAGGTTCATCTCAG (herein incorporated as SEQ ID NO: 875), found inthe 3′-UTR of ERK5 and predicted by Lewis, et al. to be bound by themir-143 miRNA, and the reporter plasmid pGL3-ERK5-3′UTR(×3) has threecontiguous copies of this sequence. The reporter plasmidpGL3-ERK5-3′UTR(ext) contains one copy of the sequenceCGGCTTGGATTATTCTGCAGGTTCATCTCAGACCCACCTTT (herein incorporated as SEQ IDNO: 876), which includes an additional ten nucleotides at either end ofthe mir-143 binding site in 3′-UTR of ERK5 predicted by Lewis, et al.(Lewis et al., Cell, 2003, 115, 787-798). The plasmidspGL3-ERK5-cds(×1), pGL3-ERK5-cds(×2), pGL3-ERK5-cds(×3), andpGL3-ERK5-cds(×5) contain one, two, three or five contiguous copies,respectively, of the novel mir-143 binding site(TTGAGCCCAGCGCTCGCATCTCA; herein incorporated as SEQ ID NO: 877) weidentified within the coding sequence of ERK5. The unmodifiedpGL3-Control luciferase reporter vector was used as a negative control,and the pGL3-mir-143 sensor reporter plasmid was used as a positivecontrol.

HeLa cells were routinely cultured and passaged as described. In someembodiments, HeLa cells were transfected with 0.05 μg of the relevantpGL3-sensor plasmid and 0.01 μg pRL-CMV plasmid. Additionally, in someembodiments, cells were treated at various dosages (11 nM, 33 nM, and100 nM) with the following oligomeric compound mimics: 1) ds-mir-143, or2) ds-Control as described. In accordance with methods described inExample 12, a luciferase assay was performed 24-hours aftertransfection. The results, shown in Tables 40 and 41, are an average ofthree trials. Data are presented as percent untreated control(luciferase plasmid only, not treated with oligomeric compound)luciferase expression, normalized to pRL-CMV levels.

TABLE 40 Effects of oligomeric compounds mimicking mir-143 on luciferaseexpression Reporter ds-mir-143 ds-control plasmid 11 nM 33 nM 100 nM 11nM 33 nM 100 nM pGL3-Control 90.7 94.2 72.5 113.4 79.6 87.0 pGL3- 50.735.4 17.2 111.3 82.6 84.7 bulge(x3) pGL3-ERK5- 81.9 84.7 62.2 103.2 79.677.6 3′UTR(x1)

From these data, it was observed that, while treatment of HeLa cellsexpressing the reporter plasmids with the ds-control did not appear tosignificantly affect luciferase expression, the mir-143 dsRNA mimiccompound inhibited luciferase activity from the pGL3-bulge(×3) sensorplasmid in a dose-dependent manner.

TABLE 41 Effects of oligomeric compounds mimicking mir-143 on luciferaseexpression ds-mir-143 ds-control 11 100 11 100 Reporter plasmid nM 33 nMnM nM 33 nM nM pGL3-Control 110.2 124.3 92.3 114.1 95.6 103.0pGL3-mir-143 sensor 15.0 15.0 11.1 114.5 108.9 97.1 pGL3-bulge(x3) 36.133.9 22.2 109.5 103.2 92.4 pGL3-ERK5-3′UTR(x1) 92.2 108.1 81.9 106.299.6 90.1 pGL3-ERK5-3′UTR(x3) 51.7 51.0 28.2 104.6 103.4 95.7pGL3-ERK5-cds(x1) 101.3 115.4 77.4 100.6 102.1 96.2 pGL3-ERK5-cds(x2)92.7 113.8 63.6 111.3 99.2 90.4 pGL3-ERK5-cds(x3) 73.5 77.9 49.4 105.296.6 79.9 pGL3-ERK5-cds(x5) 49.4 44.5 23.9 103.0 113.4 89.9pGL3-ERK5-3′UTR(ext) 89.0 106.7 81.4 96.8 108.9 89.4

From these data it was observed that treatment of HeLa cells expressingthe pGL3-bulge(×3) reporter plasmid with the ds-mir-143 miRNA mimicoligomeric compound resulted in a dose-dependent inhibition ofluciferase activity while the ds-control oligomeric compound had noeffect as described previously. Treatment of HeLa cells expressing thepGL3-ERK5-3′UTR(×1) (containing one copy of the mir-143 binding sitepredicted by Lewis, et al.) with the ds-mir-143 mimic oligomericcompound did not inhibit luciferase activity, although increasing thenumber of potential mir-143 binding sites in the pGL3-ERK5-3′UTR(×3)reporter plasmid to three appeared to favor the binding of theds-mir-143 mimic and inhibition of luciferase activity. Treatment ofcells expressing the pGL3-ERK5-cds(×1) or pGL3-ERK5-cds(×2) reporterplasmids bearing a one or two copies, respectively, of the novel mir-143binding site identified in the coding sequence of the ERK5 gene with 11-or 33 nM of the ds-mir-143 mimic oligomeric compound did not appear toinhibit luciferase activity, although treatment with 100 nM of theds-mir-143 mimic did reduce luciferase expression. Treatment of cellsexpressing the pGL3-ERK5-cds(×3) or pGL3-ERK5-cds(×5) reporter plasmids,bearing three or five of copies, respectively, of the novel mir-143binding site in the ERK5 coding sequence, with the ds-mir-143 mimicoligomeric compound resulted in a reduction in luciferase activity. ThepGL3-ERK5-cds(×5) reporter plasmid exhibited a dose-responsiveness withincreasing concentration of the mir-143 mimic oligomeric compound. Takentogether, these results support the conclusion that multiple miRNAs andmiRNA binding sites may cooperate to silence gene expression.

In order to assess the ability of miRNAs to bind predicted miRNA bindingsites and regulate the expression of the luciferase reporter, in someembodiments, expression systems based on the pGL3-Control (PromegaCorp., Madison Wis.) reporter vector and comprising either a mir-15a,mir-21, or a mir-23b miRNA binding site were developed and used intransient transfections of HeLa cells to determine whether theendogenous mir-15a, mir-21, or mir-23b miRNAs, respectively, couldrepress luciferase reporter gene expression.

The pGL3-mir-15a sensor plasmid was created by cloning the sequence(CACAAACCATTATGTGCTGCTA; SEQ ID NO: 369), complementary to the mir-15amiRNA, into the Xba site of the pGL3-Control plasmid, placing thepotential miRNA-binding site in the 3′UTR of the luciferase reporter.This reporter plasmid was used to transfect HeLa cells and it wasobserved that the endogenous mir-15a miRNA was able to inhibitluciferase expression from the pGL3-mir-15a sensor plasmid. Thus, tofurther evaluate the ability of the mir-15a miRNA to bind this targetsite encoded by the pGL3-mir-15a sensor plasmid, and to assess theability of oligomeric compounds to interfere with mir-15a-mediatedsilencing, pGL3-mir-15a sensor-expressing HeLa cells were treated withvarying concentrations (3-, 10- or 30 nM) of the following oligomericcompounds: ISIS Number 327951 (SEQ ID NO: 369) is a uniform 2′-MOEcompound targeting the mature mir-15a-1 miRNA. ISIS Numbers 356213 (SEQID NO: 878), 356215 (SEQ ID NO: 879), 356216 (SEQ ID NO: 880), 356218(SEQ ID NO: 881), 356221 (SEQ ID NO: 882), 356227 (SEQ ID NO: 883) and356229 (SEQ ID NO: 884) are phosphorothioate, uniform 2′-MOE oligomericcompounds designed and synthesized to target the entire length of themir-15a pri-miRNA molecule (described in detail in Example 28, below).The uniform 2′-MOE phosphorothioate oligomeric compounds ISIS Number327901 (SEQ ID NO: 319), targeting an unrelated miRNA (mir-143) and ISISNumber 342673 (AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO:758), containing 15 mismatches with respect to the mature mir-143 miRNA,were used as negative controls. The data presented in Table 42 are theaverage of three trials and are presented as percent untreated control(luciferase plasmid only, not treated with oligomeric compound)luciferase expression, normalized to pRL-CMV levels.

TABLE 42 Effects of oligomeric compounds on mir-15a miRNA-mediatedinhibition of luciferase expression Relative luciferase activity Dose ofSEQ ID oligomeric compound Treatment NO 3 nM 10 nM 30 nM 327901 319 83.696.6 88.2 negative control 342673 758 104.5 82.6 85.7 negative control327951 369 151.0 207.6 137.1 356213 878 101.2 80.5 109.9 356215 879 98.0116.7 79.6 356216 880 102.8 84.7 113.2 356218 881 91.6 110.3 85.7 356221882 106.8 74.0 81.2 356227 883 86.1 117.8 101.5 356229 884 109.7 100.397.5

From these data, it was observed that the oligomeric compound ISISNumber 327951 targeting the mature mir-15a miRNA blocked the inhibitoryeffect of mir-15a, exhibited as a recovery and increase in luciferaseactivity in HeLa cells expressing the pGL3-mir-15a sensor plasmid.

The pGL3-mir-23b sensor plasmid was created by cloning the sequence(GTGGTAATCCCTGGCAATGTGAT; SEQ ID NO: 307), representing a sequencecomplementary to the mir-23b miRNA, into the Xba site of thepGL3-Control plasmid, placing the potential miRNA-binding site in the3′UTR of the luciferase reporter. This reporter plasmid was used totransfect HeLa cells and it was observed that the endogenous mir-23bmiRNA was able to inhibit luciferase expression from the pGL3-mir-23bsensor plasmid. Thus, to further evaluate the ability of the mir-23bmiRNA to bind this target site encoded by the pGL3-mir-23b sensorplasmid, and to assess the ability of oligomeric compounds to interferewith mir-23b-mediated silencing, pGL3-mir-23b sensor-expressing HeLacells were treated with varying concentrations (1.3-, 5- or 20 nM) ofthe following oligomeric compounds: ISIS Number 327889 (SEQ ID NO: 307),a phosphorothioate uniform 2′-MOE oligomeric compound, and ISIS Number340925 (SEQ ID NO: 307), a 2′-MOE 5-10-8 gapmer oligomeric compound,both targeting mir-23b. The uniform 2′-MOE phosphorothioate oligomericcompound ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelatedmiRNA (mir-129-2) was used as a negative control. The data are theaverage of three trials, and are presented in Table 43 as relativeluciferase activity (normalized to pRL-CMV luciferase plasmid only, nottreated with oligomeric compound).

TABLE 43 Effects of oligomeric compounds on mir-23b miRNA-mediatedinhibition of luciferase expression Fold change luciferase Dose of SEQID oligomeric compound Treatment NO 1.3 nM 5 nM 20 nM 327924 342 1.150.68 0.92 negative control 327889-uniform MOE 307 3.75 3.46 7.40340925-gapmer 307 0.99 1.41 1.19

From these data, it was observed that, at all doses, ISIS Number 327889,the uniform 2′-MOE oligomeric compound targeting the mature mir-23bmiRNA, de-repressed the expression of the luciferase reporter. Thus,ISIS 327889 reversed the silencing effect of the mir-23b miRNA,apparently by inhibiting the binding of mir-23b to its target siteencoded by the pGL3-mir-23b sensor plasmid.

The pGL3-mir-21 sensor plasmid was created by cloning the sequence(TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), representing a sequencecomplementary to the mir-21 miRNA, into the Xba site of the pGL3-Controlplasmid, placing the potential miRNA-binding site in the 3′UTR of theluciferase reporter. This reporter plasmid was used to transfect HeLacells and it was observed that the endogenous mir-21 miRNA was able toinhibit luciferase expression from the pGL3-mir-21 sensor plasmid. Thus,to further evaluate the ability of the mir-21 miRNA to bind this targetsite encoded by the pGL3-mir-21 sensor plasmid, and to assess theability of oligomeric compounds to interfere with mir-21-mediatedsilencing, pGL3-mir-21 sensor-expressing HeLa cells were treated withvarying concentrations (10 nM or 50 nM) of the following oligomericcompounds: ISIS Number 327917 (SEQ ID NO: 335), a phosphorothioateuniform 2′-MOE oligomeric compound; ISIS Number 338697(TGCCATGAGATTCAACAGTC; herein incorporated as SEQ ID NO: 524), a uniform2′-MOE oligomeric compound targeting the mir-21 pri-miRNA molecule; andISIS Number 328415 (SEQ ID NO: 524), a 2′-MOE 5-10-5 gapmer oligomericcompound targeting the mir-21 pri-miRNA. The uniform 2′-MOEphosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319)targeting an unrelated miRNA (mir-143) was used as a negative control.The data are the average of three trials and are presented in Table 44as percent untreated control (luciferase plasmid only, not treated witholigomeric compound) luciferase expression, normalized to pRL-CMVlevels.

TABLE 44 Effects of oligomeric compounds on mir-21 miRNA-mediatedinhibition of luciferase expression % UTC Dose of oligomeric SEQ IDcompound Treatment NO 10 nM 50 nM 327901 319 74.2 83.1 negative control327917 335 1037.6 847.5 338697 524 87.0 84.8 328415 524 66.0 104.4

From these data, it was observed that, at both doses, treatment of HeLacells with ISIS Number 327917, the uniform 2′-MOE oligomeric compoundtargeting the mature mir-21 miRNA, de-repressed the expression of theluciferase reporter. Thus, ISIS 327917 reversed the silencing effect ofthe endogenous mir-21 miRNA, apparently by inhibiting the binding ofmir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.

Therefore, oligomeric compounds targeting and/or mimicking the mir-143,mir-15a, mir-23b and mir-21 miRNAs and their corresponding pri-miRNAmolecules have been demonstrated to bind to target RNA transcripts andsilence reporter gene expression.

Example 28 Effects of Oligomeric Compounds on Expression of pri-miRNAs

As described above in Example 19, pri-miRNAs, often hundreds ofnucleotides in length, are processed by a nuclear enzyme in the RNaseIII family known as Drosha, into approximately 70 nucleotide-longpre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs orfoldback miRNA precursors), and pre-miRNAs are subsequently exportedfrom the nucleus to the cytoplasm, where they are processed by humanDicer into double-stranded miRNAs, which are subsequently processed bythe Dicer RNase into mature miRNAs. It is believed that, in processingthe pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNAat the base of the mature miRNA, leaving a 2-nt 3′overhang (Lee, et al.,Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, asignature of RNaseIII cleavage, has been identified as a criticalspecificity determinant in targeting and maintaining small RNAs in theRNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol.,2004, 16, 223-9).

The oligomeric compounds of the present invention are believed todisrupt pri-miRNA and/or pre-miRNA structures, and sterically hinderDrosha and/or Dicer cleavage, respectively. Additionally, oligomericcompounds capable of binding to the mature miRNA are believed to preventthe RISC-mediated binding of a miRNA to its mRNA target, either bycleavage or steric occlusion of the miRNA.

Using the real-time RT-PCR methods described in Example 19, theexpression levels of the mir-15a pri-miRNA were compared in HepG2 cellstreated with a nested series of chimeric gapmer oligomeric compounds,targeting and spanning the entire length of the mir-15a pri-miRNA; thesecompounds are shown in Table 45, below. Each gapmer is 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings.” The wings are composed of2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. Using the transfection methodsdescribed herein, HepG2 cells were treated with 100 nM of each of thesegapmer oligomeric compounds. Total RNA was isolated from HepG2 cells bylysing cells in 1 mL TRIZOL™ (Invitrogen) using the manufacturer'srecommended protocols. Real-time RT-PCR analysis was performed using aprimer/probe set specific for the mir-15a pri-miRNA molecule to assessthe effects of these compounds on expression of the mir-15a pri-miRNAmolecule. ISIS 339317 (GTGTGTTTAAAAAAAATAAAACCTTGGA; SEQ ID NO.: 885)was used as the forward primer, ISIS 339318 (TGGCCTGCACCTTTTCAAA; SEQ IDNO.: 886) was used as the reverse primer, and ISIS 339319(AAAGTAGCAGCACATAATGGTTTGTGG; SEQ ID NO.: 887) was used as the probe.Total RNA was quantified using RiboGreen™ RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.), expression levels observed foreach target are normalized to 5.8S rRNA, and values are expressedrelative to the untreated control. Inhibition of expression of themir15a pri-miRNA by these gapmer oligomeric compounds is expressed as apercentage of RNA levels in untreated control cells. Results of theseexperiments are described in Table 45 below:

TABLE 45 Effects of chimeric oligomeric compounds on expression of themir-15a pri-miRNA SEQ Expression of ISIS ID mir-15a pri-miRNA Number NOSequence (% UTC) 347964 878 TATAACATTGATGTAATATG 13.7 347965 888GCTACTTTACTCCAAGGTTT 86.0 347966 879 TGCTACTTTACTCCAAGGTT 39.2 347967880 GCACCTTTTCAAAATCCACA 152.3 347968 889 CCTGCACCTTTTCAAAATCC 8.4347969 881 TGGCCTGCACCTTTTCAAAA 39.5 347970 890 ATATGGCCTGCACCTTTTCA 2.2347971 891 ACAATATGGCCTGCACCTTT 92.8 347972 882 AGCACAATATGGCCTGCACC98.6 347973 892 GGCAGCACAATATGGCCTGC 143.3 347974 893TGAGGCAGCACAATATGGCC 98.1 347975 894 TTTTGAGGCAGCACAATATG 9.2 347976 895TATTTTTGAGGCAGCACAAT 73.0 347977 896 TTGTATTTTTGAGGCAGCAC 111.3 347978883 TCCTTGTATTTTTGAGGCAG 51.1 347979 897 AGATCCTTGTATTTTTGAGG 74.9347980 884 AGATCAGATCCTTGTATTTT 3.6 347981 898 AGAAGATCAGATCCTTGTAT N/D347982 899 TTCAGAAGATCAGATCCTTG 82.2 347983 900 AAATATATTTTCTTCAGAAG13.0

From these data, it was observed that oligomeric compounds ISIS Numbers347964, 347966, 347968, 347970, 347975, 347980 and 347983 showsignificant inhibition of expression of the mir-15a pri-miRNA molecule.Thus, it is believed that the antisense oligomeric compounds ISISNumbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 bindto the mir-15a pri-miRNA and/or pre-miRNA molecules and cause theirdegradation and cleavage.

From these data, it was observed that oligomeric compounds ISIS Numbers347967, 347977 and 347973 stimulate an increase in expression levels ofthe mir-15a pri-miRNA. It is believed that the oligomeric compounds ISISNumbers 347967, 347977 and 347973 bind to the mir-15a pri-miRNA andinhibit its processing into the mature mir-15a miRNA. It is believedthat, in addition to the increase in the levels of the mir-15a pri-miRNAobserved upon treatment of cells with the oligomeric compounds ISISNumbers 347977, 347967 and 347973, a drop in expression levels of thefully processed mature mir-15a miRNA may also trigger a feedbackmechanism signaling these cells to increase production of the mir-15apri-miRNA.

The gapmer oligomeric compounds targeting the mir-15b and mir-15-a-1mature miRNAs described above were also transfected into T47D cellsaccording to standard procedures. In addition, uniform 2′-MOE and 2′-MOEgapmer oligomeric compounds targeting the mature mir-15a-1 and mir-15bmiRNAs were also transfected into T47D cells, for analysis of theireffects on mir-15a-1 and mir-15b pri-miRNA levels. The oligomericcompounds ISIS Number 327927 (SEQ ID NO: 345), a uniform 2′-MOE compoundand ISIS Number 345391 (SEQ ID NO: 345), a 2′-MOE 5-10-7 gapmercompound, both target mir-15b. The oligomeric compounds ISIS Number327951 (SEQ ID NO: 369), a uniform 2′-MOE compound, and ISIS Number345411 (SEQ ID NO: 369), a 2′-MOE 5-10-7 gapmer compound, both targetmir-15a-1. Oligomeric compounds ISIS Number 129686(CGTTATTAACCTCCGTTGAA; SEQ ID NO: 901), and ISIS Number 129691(ATGCATACTACGAAAGGCCG; SEQ ID NO:902), both universal scrambledcontrols, as well as ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ IDNO: 844) targeting an unrelated gene, PTEN, were used as negativecontrols. ISIS Numbers 129686, 129691, and 116847 are phosphorothiated2′-MOE 5-10-5 gapmers, and all cytosines are 5-methylcytosines. T47Dcells (seeded in 12-well plates) were treated with these oligomericcompounds, and RNA was isolated from the treated cells by lysing in 1 mLTRIZOL™ (Invitrogen) and total RNA was prepared using the manufacturer'srecommended protocols. To assess the effects of these compounds onexpression of the mir-15a or mir-15b pri-miRNA molecules, real-timeRT-PCR analysis was performed using either the primer/probe set specificfor the mir-15a pri-miRNA molecule described above, or a primer probeset specific for the mir-15b pri-miRNA molecule: ISIS 339320(CCTACATTTTTGAGGCCTTAAAGTACTG; SEQ ID NO: 903) was used as the forwardprimer for the mir-15b pri-miRNA, ISIS 339321 (CAAATAATGATTCGCATCTTGACTGT; SEQ ID NO: 904) was used as the reverse primer for the mir-15bpri-miRNA, and ISIS 339322 (AGCAGCACATCATGGTTTACATGC; SEQ ID NO: 905)was used as the probe. Total RNA was quantified using RiboGreen™ RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.),expression levels observed for each target were normalized to 5.8S rRNA,and values are expressed relative to the untreated control. Inhibitionof expression of the mir15a or mir-15b pri-miRNA molecules upontreatment with these oligomeric compounds is was assessed and expressedas a percentage of RNA levels in untreated control cells.

On multiple repeats of these experiments, it was observed that theuniform 2′-MOE oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345)and ISIS Number 327951 (SEQ ID NO: 369), targeted to the mature mir-15band mir-15a-1 miRNAs, respectively, each stimulate an approximately 2.5-to 3.5-fold increase in expression of the mir-15a pri-miRNA molecule andan approximately 1.5- to 2.5-fold increase in the expression of themir-15b pri-miRNA molecule. Therefore, it is believed that ISIS Numbers327927 and 327951 can bind to the mir-15a and/or mir-15b pri-miRNA orpre-miRNA molecules and interfere with their processing into the maturemir-15a or mir-15b miRNAs. It is also recognized that a decrease inlevels of the mature, processed forms of the mir-15a or mir-15b miRNAsin T47D cells treated with ISIS Number 345411 (SEQ ID NO: 369), ISISNumber 327927 (SEQ ID NO: 345) or ISIS Number 327951 (SEQ ID NO: 369)may also trigger a feedback mechanism that signals these cells toincrease production of the mir-15a and/or mir-15b pri-miRNA molecules.

In accordance with the present invention, a nested series of uniform2′-MOE oligomeric compounds were designed and synthesized to target theentire length of the mir-15a pri-miRNA molecule. Each compound is 19nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotidesand phosphorothioate (P═S) internucleoside linkages throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds are shown in Table 46. The compounds can be analyzed for theireffect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitativereal-time PCR, or they can be used in other assays to investigate therole of miRNAs or the function of targets downstream of miRNAs.

TABLE 46 Uniform 2′-MOE PS Compounds targeting the mir-15a pri-miRNAISIS SEQ ID Number NO Sequence 356213 878 TATAACATTGATGTAATATG 356214879 GCTACTTTACTCCAAGGTTT 356215 880 TGCTACTTTACTCCAAGGTT 356216 881GCACCTTTTCAAAATCCACA 356217 882 CCTGCACCTTTTCAAAATCC 356218 883TGGCCTGCACCTTTTCAAAA 356219 884 ATATGGCCTGCACCTTTTCA 356220 888ACAATATGGCCTGCACCTTT 356221 889 AGCACAATATGGCCTGCACC 356222 890GGCAGCACAATATGGCCTGC 356223 891 TGAGGCAGCACAATATGGCC 356224 892TTTTGAGGCAGCACAATATG 356225 893 TATTTTTGAGGCAGCACAAT 356226 894TTGTATTTTTGAGGCAGCAC 356227 895 TCCTTGTATTTTTGAGGCAG 356228 896AGATCCTTGTATTTTTGAGG 356229 897 AGATCAGATCCTTGTATTTT 356230 898AGAAGATCAGATCCTTGTAT 356231 899 TTCAGAAGATCAGATCCTTG 356232 900AAATATATTTTCTTCAGAAG

Using the real-time RT-PCR methods described, the expression levels ofthe mir-15a pri-miRNA were compared in T47D cells treated with thenested series of uniform 2′-MOE oligomeric compounds, targeting andspanning the entire length of the mir-15a pri-miRNA. The regionencompassing the mir-15a primary transcript (the complement ofnucleotides 31603159 to 31603468 of GenBank Accession numberNT_(—)024524.13; AAATAATTATGCATATTACATCAATGTTATAATGTTTAAACATAGATTTTTTTACATGCATTCTTTTTTTCCTGAAAGAAAATATTTTTTATATTCTTTAGGCGCGAATGTGTGTTTAAAAAAAATAAAACCTTGGAGTAAAGTAGCAGCACATAATGGTTTGTGGATTTTGAAAAGGTGCAGGCCATATTGTGCTGCCTCAAAAATACAAGGATCTGATCTTCTGAAGAAAATATATTTCTTTTTATTCATAGCTCTTATGATAGCAATGTCAGCAGTGCCTTAGCAGCACGTAAATATTGGCGTTAAG) isincorporated herein as SEQ ID NO: 906. ISIS Number 356215 (SEQ ID NO:879) targets a region flanking and immediately 5′ to the predicted 5′Drosha cleavage site in the mir-15a pri-miRNA. ISIS Number 356218 (SEQID NO: 881) targets a region in the loop of the mir-15a pri-miRNA. ISIS356227 (SEQ ID NO: 883) targets a region flanking and immediately 3′ tothe predicted 3′ Drosha cleavage site in the mir-15a pri-miRNA.Additionally, oligomeric compound ISIS 327951 (SEQ ID NO: 369), auniform 2′-MOE compound targeting the mature mir-15a-1 miRNA, was testedfor comparison. Oligomeric compounds ISIS 327901 (SEQ ID NO: 319)targeting the mature mir-143 miRNA; ISIS 129690, (TTAGAATACGTCGCGTTATG;SEQ ID NO: 907), a phosphorothioate 5-10-5 MOE gapmer used as auniversal scrambled control; and ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQID NO: 844), a uniform 5-10-52′-MOE gapmer targeting an unrelated gene,PTEN, were used as negative controls. Using the transfection methodspreviously described, T47D cells were treated with 100 nM of each ofthese oligomeric compounds. Total RNA was isolated by lysing cells in 1mL TRIZOL™ (Invitrogen) using the manufacturer's recommended protocols.real-time RT-PCR analysis was performed using a primer/probe setspecific for the mir-15a pri-miRNA molecule [forward primer-ISIS 339317(SEQ ID NO.: 885), reverse primer-ISIS 339318 (SEQ ID NO.: 886), andprobe=ISIS 339319 (SEQ ID NO.: 887)]. Total RNA was quantified usingRiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.), expression levels observed for each target were normalized to5.8S rRNA, and values were expressed relative to the untreated control(UTC). Effects on expression of the mir-15a pri-miRNA molecule resultingfrom treatment of T47D cells with these uniform 2′-MOE oligomericcompounds is expressed as a percentage of RNA levels in untreatedcontrol cells. Results of these experiments are described in Table 47below:

TABLE 47 Effects of uniform 2′-MOE oligomeric compounds on mir-15apri-miRNA expression ISIS # SEQ ID NO: target % UTC UTC N/A N/A 100129690 XXX N/A 121 scrambled control 327901 319 mir-143 132 116847 844PTEN mRNA 132 327951 369 mature mir-15a-1 713 356213 878 >100 bpupstream of mature mir-15a 171 356215 879 flanking 5′ Drosha cleavagesite 1005 of mir-15a-1 pri-miRNA 356216 880 mir-15a-1 pri-miRNA 503356218 881 loop of mir-15a-1 pri-miRNA 392 356221 882 mir-15a-1pri-miRNA 444 356224 894 mir-15a-1 pri-miRNA 592 356227 883 flanking 3′Drosha cleavage site 879 of mir-15a-1 pri-miRNA 356229 884 mir-15a-1pri-miRNA 818 356231 899 mir-15a-1 pri-miRNA 811 356232 900 mir-15a-1pri-miRNA 631

From these data, it was observed that the uniform 2′-MOE oligomericcompounds ISIS Numbers 327927, 327951, 356215, 356216, 356218, 356221,356224, 356227, 356229, 356231 and 356232 stimulate an increase inlevels of the mir-15a pri-miRNA molecule as detected by real-timeRT-PCR. Notably, oligomeric compounds ISIS Numbers 356215 and 356227which target the regions immediately flanking the predicted 5′ and 3′Drosha cleavage sites in the mir-15a pri-miRNA, respectively, wereobserved to stimulate the greatest increases in expression of themir-15a pri-miRNA. It is believed that these oligomeric compounds bindto the mir-15a pri-miRNA and/or pre-miRNA molecules and interfere withtheir processing into the mature mir-15a miRNA, possibly by interferingwith the activity of RNase III-like enzymes such as human Dicer and/orDrosha. The resultant decrease in levels of the processed mature mir-15amiRNA may trigger a feedback mechanism leading to an upregulation ofproduction of the mir-15a pri-miRNA molecule. Not mutually exclusivewith the processing interference and the feedback mechanisms is thepossibility that treatment with oligomeric compounds could stimulate theactivity of an RNA-dependent RNA polymerase (RdRP) that amplifies themir-15a pri-miRNA or pre-miRNA molecules. It is understood that sucholigomeric compound-triggered mechanisms may be operating not only uponregulation of mir-15a production and processing, but may also be foundto regulate the production and processing of other miRNAs.

The expression levels of mir-24-2, let-7i, and let-7d were assessed inHeLa or T-24 cells treated with various uniform 2′-MOE oligomericcompounds targeting mature miRNAs. For example, using the transfectionmethods previously described, HeLa cells were treated with 100 nM of theoligomeric compound ISIS Number 327945 (SEQ ID NO: 363) targeting themir-24-2 mature miRNA. Total RNA was isolated and expression levels ofthe mir-24-2 pri-miRNA were analyzed by real-time quantitative RT-PCRusing a primer/probe set specific for the mir-24-2 pri-miRNA molecule[forward primer=ISIS 359358 (CCCTGGGCTCTGCCT; herein incorporated as SEQID NO.: 908), reverse primer-ISIS 359359 (TGTACACAAACCAACTGTGTTTC;herein incorporated as SEQ ID NO.: 909), and probe=ISIS 359360(CGTGCCTACTGAGC; herein incorporated as SEQ ID NO.: 910)]. Anapproximately 35-fold increase in expression levels of the mir-24-2pri-miRNA molecule was observed in HeLa cells treated with theoligomeric compound ISIS 327945 as detected by real-time RT-PCR.

Using the transfection methods previously described, HeLa cells weretreated with 100 nM of the oligomeric compound ISIS Number 327890 (SEQID NO: 308) targeting the let-7i mature miRNA. Total RNA was isolatedand expression levels of the let-7i pri-miRNA were analyzed by real-timequantitative RT-PCR using a primer/probe set specific for the let-7ipri-miRNA molecule [forward primer-ISIS 341684(TGAGGTAGTAGTTTGTGCTGTTGGT; herein incorporated as SEQ ID NO.: 777),reverse primer=ISIS 341685 (AGGCAGTAGCTTGCGCAGTTA; herein incorporatedas SEQ ID NO.: 778), and probe=ISIS 341686 (TTGTGACATTGCCCGCTGTGGAG;herein incorporated as SEQ ID NO.: 779)]. An approximately 4-foldincrease in expression levels of the let-7i pri-miRNA molecule wasobserved in HeLa cells treated with the oligomeric compound ISIS 327890as detected by real-time RT-PCR.

Using the transfection methods previously described, supra, T-24 cellswere treated with 100 nM of the oligomeric compound ISIS Number 327926(SEQ ID NO: 344) targeting the let-7d mature miRNA. Total RNA wasisolated and expression levels of the let-7d pri-miRNA were analyzed byreal-time quantitative RT-PCR using a primer/probe set specific for thelet-7d pri-miRNA molecule (forward primer=ISIS 341678 (CCTAGGAAGAGGTAGTAGGTTGCA; herein incorporated as SEQ ID NO.: 771), reverse primer=ISIS341679 (CAGCAGGTCGTATAGTTACCTCCTT; herein incorporated as SEQ ID NO.:772), and probe=ISIS 341680 (AGTTTTAGGGCAGGGATTTTGCCCA; hereinincorporated as SEQ ID NO.: 773)). An approximately 1.7-fold increase inexpression levels of the let-7d pri-miRNA molecule was observed in T-24cells treated with the oligomeric compound ISIS 327926 as detected byreal-time RT-PCR.

Thus, treatment with uniform 2′-MOE oligomeric compounds targetingmature miRNAs appears to result in an induction of expression of thecorresponding pri-miRNA molecule.

In one embodiment, the expression of mir-21 (noted to be expressed athigh levels in HeLa cells) was assessed in cells treated with oligomericcompounds. Using the transfection methods previously described, HeLacells were treated with 100 nM of the uniform 2′-MOE oligomeric compoundISIS Number 327917 (SEQ ID NO: 335) targeting the mir-21 mature miRNA.Total RNA was isolated by lysing cells in 1 mL TRIZOL™ (Invitrogen)using the manufacturer's recommended protocols. By Northern blotanalysis of total RNA from HeLa cells treated with ISIS 327917,expression levels of the mir-21 mature miRNA were observed to be reducedto 50% of those of untreated control cells. Furthermore, expressionlevels of the mir-21 pri-miRNA were found to increase in these HeLacells treated with the oligomeric compound ISIS 327917. Real-time RT-PCRanalysis was also performed on HeLa cells treated with ISIS 327917 usinga primer/probe set specific for the mir-21 pri-miRNA molecule [forwardprimer=ISIS 339332 (GCTGTACCACCTTGTCGGGT; herein incorporated as SEQ IDNO.: 911), reverse primer-ISIS 339333 (TCGACTGGTGTTGCCATGA; hereinincorporated as SEQ ID NO.: 912), and probe=ISIS 339334(CTTATCAGACTGATGTTGACTGTTGAAT; herein incorporated as SEQ ID NO.: 913)].Total RNA was quantified using RiboGreen™ RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.), expression levels observed forthe target were normalized to 5.8S rRNA, and values were expressedrelative to an untreated control (UTC). ISIS Number 327917 was observedto stimulate an approximately 2-fold increase in levels of the mir-21pri-miRNA molecule as detected by real-time RT-PCR.

Thus, it is believed that, in addition to binding the mir-21 maturemiRNA and interfering with the RISC-mediated binding of mir-21 to itsmRNA target, the oligomeric compound, ISIS 327917, binds to the mir-21pri-miRNA and/or pre-miRNA molecules and interferes with theirprocessing into the mature mir-21 miRNA, inhibiting expression of themature mir-21 miRNA in HeLa cells, possibly by interfering with theactivity of RNase III-like enzymes such as human Dicer or Drosha. Theresultant decrease in levels of mature mir-21 miRNA may trigger afeedback mechanism leading to an upregulation of production of themir-21 pri-miRNA molecule. Treatment with this oligomeric compound couldalso stimulate the activity of an RNA-dependent RNA polymerase (RdRP)that amplifies the mir-21 pri-miRNA or pre-miRNA molecules.

In accordance with the present invention, a nested series of uniform2′-MOE oligomeric compounds were designed and synthesized to target theentire length of the mir-21 pri-miRNA molecule. The region encompassingthe mir-21 primary transcript (nucleotides 16571584 to 16571864 ofGenBank Accession number NT_(—)010783.14;CTGGGTTTTTTTGGTTTGTTTTTGTTTTTGTTTTTTTATCAAATCCTGCCTGACTGTCTGCTTGTTTTGCCTACCATCGTGACATCTCCATGGCTGTACCACCTTGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGACATTTTGGTATCTTTCATCTGACCATCCATATCCAATGTTCTCATTTAAACATTACCCAGCATCATTGTTTATAATCAGAAACTCTGGTCCTTCTGTCTGGTGGCAC) is incorporated herein as SEQ IDNO: 914. Each compound is 20 nucleotides in length, composed of2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S)internucleoside linkages throughout the compound. All cytidine residuesare 5-methylcytidines. The compounds are shown in Table 48. Thecompounds can be analyzed for their effect on mature miRNA, pre-miRNA orpri-miRNA levels by quantitative real-time PCR, or they can be used inother assays to investigate the role of miRNAs or the function oftargets downstream of miRNAs.

TABLE 48 Uniform 2′-MOE PS Compounds targeting the mir-21 pri-miRNA ISISSEQ ID Number NO Sequence 358765 915 ACAAGCAGACAGTCAGGCAG 358766 916GGTAGGCAAAACAAGCAGAC 358767 917 GGAGATGTCACGATGGTAGG 358768 918AGGTGGTACAGCCATGGAGA 358769 919 GATAAGCTACCCGACAAGGT 358770 920AGTCTGATAAGCTACCCGAC 358771 921 CAACAGTCAACATCAGTCTG 358772 922GAGATTCAACAGTCAACATC 358773 923 CTGGTGTTGCCATGAGATTC 358774 924CATCGACTGGTGTTGCCATG 358775 925 ACAGCCCATCGACTGGTGTT 358776 926TGTCAGACAGCCCATCGACT 358777 927 CCAAAATGTCAGACAGCCCA 358778 928GATACCAAAATGTCAGACAG 358779 929 GGTCAGATGAAAGATACCAA 358780 930AACATTGGATATGGATGGTC 358781 931 TAATGTTTAAATGAGAACAT 358782 932AACAATGATGCTGGGTAATG 358783 933 GAGTTTCTGATTATAAACAA 358784 934CGACAAGGTGGTACAGCCAT 358785 935 GAAAGATACCAAAATGTCAG

Using the real-time RT-PCR methods, the expression levels of the mir-21pri-miRNA were compared in HeLa cells treated with this nested series ofuniform 2′-MOE oligomeric compounds, targeting and spanning the entirelength of the mir-21 pri-miRNA. ISIS Number 358768 (SEQ ID NO: 918)targets a region flanking the predicted 5′ Drosha cleavage site in themir-21 pri-miRNA. ISIS Number 358777 (SEQ ID NO: 927) targets a regionspanning the 3′ Drosha cleavage site in the mir-21 pri-miRNA. ISIS358779 (SEQ ID NO: 929) targets a region flanking the predicted 3′Drosha cleavage site in the mir-21 pri-miRNA. Additionally, oligomericcompounds ISIS 327917 (SEQ ID NO: 335), a uniform 2′-MOE compoundtargeting the mature mir-21 miRNA, and ISIS 345382(TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), a 5-10-7 phosphorothioate2′-MOE gapmer targeting mir-21, were tested for comparison. Oligomericcompound ISIS 327863 (ACGCTAGCCTAATAGCGAGG; herein incorporated as SEQID NO: 936), a phosphorothioate 5-10-52′-MOE gapmer, was used asscrambled control. Using the transfection methods previously described,HeLa cells were treated with 100 nM of each of these oligomericcompounds. Total RNA was isolated by lysing cells in 1 mL TRIZOL™(Invitrogen) using the manufacturer's recommended protocols. real-timeRT-PCR analysis was performed using the primer/probe set specific forthe mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (SEQ ID NO.:911), reverse primer-ISIS 339333 (SEQ ID NO.: 912), and probe=ISIS339334 (SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreen™ RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.),expression levels observed for each target were normalized to 5.8S rRNA,and values were expressed relative to the untreated control (UTC).Effects on expression of the mir-21 pri-miRNA molecule resulting fromtreatment of HeLa cells with these uniform 2′-MOE oligomeric compoundsis expressed as a percentage of RNA levels in untreated control cells.Results of these experiments are shown in Table 49 below:

TABLE 49 Effects of oligomeric compounds on mir-21 pri-miRNA expressionISIS # SEQ ID NO: target % UTC UTC N/A N/A 100 327863 936 N/A 107 gapmercontrol 327917 335 mature mir-21 249 uniform 2′-MOE 345382 335 maturemir-21 119 5-10-7 2′-MOE gapmer 358765 915 mir-21 pri-miRNA 133 358766916 mir-21 pri-miRNA 142 358767 917 mir-21 pri-miRNA 248 358768 918flanking 5′ Drosha cleavage site 987 of mir-21 pri-miRNA 358769 919mir-21 pri-miRNA 265 358770 920 mir-21 pri-miRNA 250 358771 921 mir-21pri-miRNA 181 358772 922 mir-21 pri-miRNA 245 358773 923 mir-21pri-miRNA 148 358774 924 mir-21 pri-miRNA 104 358775 925 mir-21pri-miRNA 222 358776 926 mir-21 pri-miRNA 367 358777 927 spanning 3′Drosha cleavage site 536 of mir-21 pri-miRNA 358778 928 mir-21 pri-miRNA503 358779 929 flanking 3′ Drosha cleavage site 646 of mir-21 pri-miRNA358780 930 mir-21 pri-miRNA 269 358781 931 mir-21 pri-miRNA 122 358782932 mir-21 pri-miRNA 155 358783 933 mir-21 pri-miRNA 133 358784 934mir-21 pri-miRNA 358 358785 935 mir-21 pri-miRNA 257

From these data, it was observed that the uniform 2′-MOE oligomericcompounds ISIS Numbers 327917, 358767, 358768, 358769, 358770, 358772,358775, 358776, 358777, 358778, 358779, 358780, 358784 and 358785stimulate an increase in levels of the mir-21 pri-miRNA molecule asdetected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers358768 and 358779 which target the regions flanking the predicted 5′ and3′ Drosha cleavage sites, respectively, and ISIS Number 358777, whichtargets a region spanning the 3′ Drosha cleavage site in the mir-21pri-miRNA were observed to stimulate the greatest increases inexpression of the mir-21 pri-miRNA. Furthermore, treatment of HeLa cellswith increasing concentrations (25, 50, 100, and 200 nM) of ISIS Numbers358768, 358779, and 327917 was observed to result in a dose-responsiveinduction of mir-21 pri-miRNA levels. Thus, it is believed that theseoligomeric compounds bind to the mir-21 pri-miRNA and/or pre-miRNAmolecules and interfere with their processing into the mature mir-21miRNA, possibly by interfering with the activity of RNase III-likeenzymes such as human Dicer and/or Drosha. The resultant decrease inlevels of the processed mature mir-21 miRNA may trigger a feedbackmechanism leading to an upregulation of production of the mir-21pri-miRNA molecule. Not mutually exclusive with the processinginterference and the feedback mechanisms is the possibility thattreatment with oligomeric compounds could stimulate the activity of anRNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNAor pre-miRNA molecules. It is understood that such oligomericcompound-triggered mechanisms may be operating not only upon regulationof mir-21 production and processing, but may also be found to regulatethe production and processing of other miRNAs or target nucleic acids.

In one embodiment, the oligomeric compounds ISIS Number 327917 (SEQ IDNO: 335), the phosphorothioate uniform 2′-MOE targeting mature mir-21;ISIS Number 358768 (SEQ ID NO: 918), the uniform 2′-MOE targeting themir-21 pri-miRNA which stimulated the largest increase in pri-miRNAexpression levels by real time quantitative RT-PCR; and ISIS Number345382 (SEQ ID NO: 335), the 5-10-7 phosphorothioate 2′-MOE gapmertargeting mature mir-21 were selected for dose response studies in HeLacells using the luciferase reporter system described in Example 27. ISISNumber 342683 (SEQ ID NO: 790), representing the scrambled nucleotidesequence of an unrelated PTP1B antisense oligonucleotide, was used as anegative control. HeLa cells expressing the pGL3-mir-21 sensor plasmid(described in Example 27) were treated with 1.9, 5.5, 16.7, and 50 nM ofthese oligomeric compounds, to assess the ability of oligomericcompounds to interfere with endogenous mir-21-mediated silencing of thepGL3-mir-21 sensor plasmid. The data are presented in Table 50 aspercent untreated control (luciferase plasmid only, not treated witholigomeric compound) luciferase expression, normalized to pRL-CMVlevels.

TABLE 50 Effects of oligomeric compounds on mir-21 miRNA-mediatedinhibition of luciferase expression % UTC Dose of oligomeric compoundTreatment 1.9 nM 5.5 nM 16.7 nM 50 nM 342683 127 171 104 108 negativecontrol 327917 522 1293 2470 4534 358768 103 163 146 118 345382 101 135117 95

From these data, it was observed that, at all doses, treatment of HeLacells with ISIS Number 327917, the uniform 2′-MOE oligomeric compoundtargeting the mature mir-21 miRNA, de-repressed the expression of theluciferase reporter, in a dose-dependent fashion. Thus, ISIS 327917reversed the silencing effect of the endogenous mir-21 miRNA, possiblyby inhibiting the binding of mir-21 to its target site encoded by thepGL3-mir-21 sensor plasmid.

Example 29 Diseases Associated with miRNA-Containing Loci

Using the public databases Online Mendelian Inheritance in Man (OMIM)(accessible through the Internet at, for example,ftp.ncbi.nih.gov/repository/OMIM/) and LocusLink (accessible at, forexample, ftp.ncbi.nlm.nih.gov/refseq/LocusLink/), a bioinformaticanalysis was performed which allowed the prediction of miRNAs associatedwith several human diseases. First, miRNAs encoded within genes havingLocusLink identification numbers were identified, and these werecompared to tables (for example, “mim2loc,” which connects LocusLinkidentification numbers with OMIM identification numbers, as well as“genemap,” “genemap.key,” “mim-title,” and “morbidmap” tables) for theconstruction of a new database called “db 1.mdb” linking miRNAs toLocusLink and OMIM identification numbers and linking these to humandiseases.

It was observed that, beginning with 95 pri-miRNAs, a subset of 49 hadOMIM identification numbers, 48 of which were linked to OMIM names. Sixof these miRNAs were associated with specific diseased patients (some ineach category were duplicates). Thus, the majority of miRNAs with OMIMidentification numbers are not directly linked to observed diseases, butare likely to be important in pathways (such as cholesterol homeostasis)associated with diseases. Tables 51 and 52 summarize informationretrieved from these studies.

TABLE 51 miRNA genes associated with specific diseases OMIM ID: locuscontaining miRNA Disease association: 120150 collagen, type I, alpha 1/Osteogenesis imperfecta, hypothetical miRNA-144 type I, 166200 114131calcitonin receptor containing Osteoporosis, hypothetical miRNA 30postmenopausal susceptibility, 166710 605317 forkhead box P2/Speech-language disorder- hypothetical miRNA 169 1, 602081 600700 LIMdomain-containing preferred Lipoma; Leukemia, translocation partner inlipoma myeloid containing miR-28 160710 myosin, heavy polypeptide 6,Cardiomyopathy, familial cardiac muscle, alpha hypertrophic, 192600(cardiomyopathy, hypertrophic 1) containing miR-208 606157 hypotheticalprotein FLJ11729 Neurodegeneration, containing mir-103-2 pantothenatekinase- associated, 234200

The previous table shows miRNAs associated with an OMIM record that werealso associated with diseased patients.

The following table, Table 52, describes diseases or disease-relatedphenotypes found to be associated with genetic loci associated with amiRNA.

TABLE 52 miRNAs associated with disease phenotypes OMIM ID: Locuscontaining miRNA Disease association: 114131 calcitonin receptorcontaining Osteoporosis, hypothetical miRNA-30 postmenopausal,susceptibility, 166710 120150 collagen, type I, alpha 1/ Osteogenesisimperfecta, hypothetical miRNA-144 type I, 166200 138247 glutamatereceptor, ionotropic, cerebellar long-term AMPA 2/hypothetical miRNA-171depression 160710 myosin, heavy polypeptide 6, Cardiomyopathy, familialcardiac muscle, alpha hypertrophic, 192600 (cardiomyopathy,hypertrophic 1) containing miR-208 184756 sterol regulatoryelement-binding Emery-Dreifuss muscular protein-1/mir-33b dystrophy,310300; dilated cardiomyopathy (CMD1A), 115200; familial partiallipodystrophy (FPLD), 151660 300093 gamma-aminobutyric acid (GABA) Aearly-onset parkinsonism, or receptor, epsilon Waisman syndrome, 311510;and MRX3 X-linked mental retardation, 309541 305660 gamma-aminobutyricacid (GABA) A manic depressive illness, receptor, alpha 3 containingmiR- colorblindness, and G6PD 105 (Mourelatos) and miR-105-2 305915glutamate receptor, ionotrophic, complex bipolar disorder; AMPA3/hypothetical miRNA-033 drug addiction 600150 potassium largeconductance cardiovascular disease calcium-activated channel, subfamilyM, alpha member 1 containing hypothetical miRNA-172 600395 glypican 1containing miR-149 angiogenesis 600481 Sterol regulatory element bindingLDL and cholesterol transcription factor 2 containing homeostasismir-33a 600592 Minichromosome maintenance increased chromosomal loss,deficient (S. cerevisiae) 7 DNA replication and containing miR-93(Mourelatos) recombination and miR-25 and miR-94 600700 LIMdomain-containing preferred Lipoma; Leukemia, myeloid translocationpartner in lipoma containing miR-28 600758 Focal adhesion kinase, p125/oncogenesis mir-151 601009 tight junction protein 1 (zona peptic ulcerdisease and occludens 1)/hypothetical miRNA- gastric carcinoma 183601029 mesoderm specific transcript intrauterine and postnatal (mouse)homolog containing mir- growth retardation 240* (Kosik) 601698 proteintyrosine phosphatase, insulin-dependent diabetes receptor type, Npolypeptide 2 mellitus (IDDM) containing mir-153-2 601773 proteintyrosine phosphatase, insulin-dependent diabetes receptor type, Ncontaining mir- mellitus (IDDM), 222100 153-1 603576 melastatin 1containing mir-211 metastatic human melanoma 603634 ribosomal proteinL5/ colorectal cancers hypothetical miRNA 168-2 603745 slit (Drosophila)homolog 3 congenital diaphragmatic containing mir-218-2 hernia 603746slit (Drosophila) homolog 2 retinal ganglion cell axon containingmir-218-1 guidance 603803 dachshund (Drosophila) homolog cellproliferation during containing hypothetical miRNA-083 mammalianretinogenesis and pituitary development 605317 forkhead boxP2/hypothetical autism & speech-language miRNA 169 disorder-1, 602081605547 follistatin-like 1 containing systemic rheumatic diseases mir-198605575 SMC4 (structural maintenance of cell proliferation chromosomes 4,yeast)-like 1 containing mir-16-3 and mir-15b 605766 deleted inlymphocytic leukemia, B-cell chronic lymphocytic 2 containing mir-16-1and mir- leukemia 15a-1 606157 hypothetical protein FLJ11729Neurodegeneration, containing mir-103-2 pantothenate kinase- associated,234200 (3); 606160 pantothenate kinase containing pantothenate kinase-mir-107 associated neurodegeneration 606161 hypothetical proteinFLJ12899 pantothenate kinase- containing mir-103-1 associatedneurodegeneration

From these data, it was observed that several miRNAs are predicted to beassociated with human disease states. For example, several studies ofautistic disorder have demonstrated linkage to a similar region of 7q(the AUTS1 locus), leading to the proposal that a single genetic factoron 7q31 contributes to both autism and language disorders, and it hasbeen reported that the FOXP2 gene, located on human 7q31, encoding atranscription factor containing a polyglutamine tract and a forkheaddomain, is mutated in a severe monogenic form of speech and languageimpairment, segregating within a single large pedigree, and is alsodisrupted by a translocation. In one recent study, association andmutation screening analysis of the FOXP2 gene was performed to assessthe impact of this gene on complex language impairments and autism, andit was concluded that coding-region variants in FOXP2 do not underliethe AUTS1 linkage and that the gene is unlikely to play a role in autismor more common forms of language impairment (Newbury, et al., Am. J.Hum. Genet. 2002, 70, 1318-27). However, hypothetical mir-169 is alsoencoded by this same genetic locus, and it is possible that mutationsaffecting the hypothetical mir-169 miRNA could underlie the AUTS1linkage and play a role in language impairment. To this end, oligomericcompounds targeting or mimicking the mir-169 miRNA may prove useful inthe study, diagnosis, treatment or amelioration of this disease.

Example 30 Effects of Oligomeric Compounds Targeting miRNAs on InsulinSignaling and Hallmark Gene Expression in HepG2 Cells

Additional oligomeric compounds were screened in the assays described inExample 18. As stated above, insulin inhibits the expression of IGFBP-1,PEPCK-c and follistatin mRNAs.

Protocols for treatment of HepG2 cells and transfection of oligomericcompounds are as described in Example 18. Also as described in Example18, forty-four hours post-transfection, the cells in the transfectedwells were treated with either no insulin (“basal” Experiment 3 (below),for identification of insulin-mimetic compounds) or with 1 nM insulin(“insulin treated” Experiment 4 (below), for identification of insulinsensitizers) for four hours. At the same time, in both plates, cells insome of the un-transfected control wells are treated with 100 nM insulinto determine maximal insulin response. At the end of the insulin orno-insulin treatment (forty-eight hours post-transfection), total RNA isisolated from both the basal and insulin treated (1 nM) 96-well plates,and the amount of total RNA from each sample is determined using aRibogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR isperformed on all the total RNA samples using primer/probe sets for threeinsulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expressionlevels for each gene are normalized to total RNA, and values±standarddeviation are expressed relative to the transfectant only and negativecontrol oligonucleotides. The compound ISIS Number 186515(AGGTAGCTTTGATTATGTAA; SEQ ID NO: 939) is targeted to IGFBP-1 and is aphosphorothioate 5-10-5 MOE gapmer where all cytosines are5-methylcytosines, as is used as a transfection control. The oligomericcompound ISIS Number 340341 (TAGCTTATCAGACTGATGTTGA; SEQ ID NO: 236) isa uniform 2′-MOE phosphorothioate compound targeted to mir-104(Mourelatos), ISIS 340362 (GACTGTTGAATCTCATGGCA; SEQ ID NO: 937) is a5-10-5 gapmer compound also targeted to mir-104 (Mourelatos), and ISISNumber 341813 (AGACACGTGCACTGTAGA; SEQ ID NO: 938) is a uniform 2′-MOEphosphorothioate compound targeted to mir-139. Results of theseexperiments are shown in Tables 53 and 54.

TABLE 53 Experiment 3: Effects of oligomeric compounds targeting miRNAson insulin-repressed gene expression in HepG2 cells SEQ ID IGFBP-1PEPCK-c Follistatin ISIS NO: NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTCN/A N/A 100 100 100  29848 737 N/A 104 100 90 n-mer 186515 939 IGFBP-1193 70 67 328384 493 hypothetical 139 142 110 miRNA-039 328677 586hypothetical 208 145 130 miRNA-120 328685 594 mir-219 157 219 100 328691600 mir-145 105 108 93 328759 668 mir-216 356 98 266 328761 670hypothetical 118 48 91 miRNA-138 328765 674 mir-215 88 93 87 328773 682mir-15a-2 148 138 131 328779 688 hypothetical 135 123 109 mir-177 340341236 mir-104 110 129 94 (Mourelatos) 340362 937 mir-104 157 168 123(Mourelatos) 341813 938 mir-139 137 121 100

Under “basal” conditions (without insulin), treatments of HepG2 cellswith oligomeric compounds of the present invention resulting indecreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/orfollistatin marker genes indicate that the oligomeric compounds have aninsulin mimetic effect. Treatments with oligomeric compounds of thepresent invention resulting in an increase in mRNA expression levels ofthe PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that thesecompounds inhibit or counteract the normal insulin repression of mRNAexpression of these genes.

From these data, it is evident that the oligomeric compound ISIS Number328761 targeting hypothetical mir-138, for example, results in a 52%decrease in PEPCK-c mRNA, a marker widely considered to beinsulin-responsive. Thus, this oligomeric compound may be useful as apharmaceutical agent with insulin mimetic properties in the treatment,amelioration, or prevention of diabetes or other metabolic diseases.

TABLE 54 Experiment 4: Effects of oligomeric compounds targeting miRNAson insulin-sensitization of gene expression in HepG2 cells SEQ IGFBP-1PEPCK-c Follistatin ISIS NO: ID NO Pri-miRNA (% UTC) (% UTC) (% UTC) UTC(1 nm N/A N/A 100 100 100 insulin)  29848 737 N/A 92 90 95 n-mer 186515939 IGFBP-1 105 40 39 328384 493 hypothetical 102 114 121 miRNA-039328677 586 hypothetical 159 117 118 miRNA-120 328685 594 mir-219 143 184157 328691 600 mir-145 101 97 104 328759 668 mir-216 212 92 224 328761670 hypothetical 93 55 98 miRNA-138 328765 674 mir-215 94 73 97 328773682 mir-15a-2 136 93 148 328779 688 hypothetical 128 78 119 mir-177340341 236 mir-104 113 115 120 (Mourelatos) 340362 937 mir-104 129 104119 (Mourelatos) 341813 938 mir-139 117 88 102

In HepG2 cells treated with 1 nM insulin, treatments with oligomericcompounds of the present invention resulting in a decrease in mRNAexpression levels of the PEPCK-c, IGFBP-1 and/or follistatin markergenes indicate that these compounds have an insulin sensitizationeffect. Treatments with oligomeric compounds of the present inventionresulting in an increase in mRNA expression levels of the PEPCK-c,IGFBP-1 and/or follistatin marker genes indicate that these compoundsinhibit or counteract the normal insulin response of repression of mRNAexpression of these genes.

From these data, it is evident that the oligomeric compounds, ISISNumber 328761 targeting hypothetical mir-138 and ISIS Number 328765targeting mir-215, for example, were observed to result in a 45% and a27% reduction, respectively, of PEPCK-c mRNA expression, widelyconsidered to be a marker of insulin-responsiveness. Furthermore, mRNAlevels of the IGFBP-1 and follistatin genes were also reduced. Thus,these oligomeric compounds may be useful as pharmaceutical agents withinsulin-sensitizing properties in the treatment, amelioration, orprevention of diabetes or other metabolic diseases.

Example 31 Adipocyte Assay of Oligomeric Compounds

The effect of several oligomeric compounds of the present inventiontargeting miRNA target nucleic acids on the expression of markers ofcellular differentiation was examined in differentiating adipocytes.

As described in Example 13, some genes known to be upregulated duringadipocyte differentiation include HSL, aP2, Glut4 and PPARγ. These genesplay important roles in the uptake of glucose and the metabolism andutilization of fats. An increase in triglyceride content is anotherwell-established marker for adipocyte differentiation.

For assaying adipocyte differentiation, expression of the four hallmarkgenes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG)accumulation were measured as previously described in adipocytestransfected with uniform 2′-MOE or chimeric gapmer phosphorothioate (PS)oligomeric compounds. Triglyceride levels as well as mRNA levels foreach of the four adipocyte differentiation hallmark genes are expressedas a percentage of untreated control (UTC) levels. Results are shown inTable 55.

TABLE 55 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers ISIS Number SEQ ID NO TG HSL AP2Glut4 PPAR gamma UTC N/A 100 100 100 100 100 ISIS-29848 737 89 84 89 96100 n-mer 327877 295 109 82 77 119 85 327888 306 132 134 102 84 103327904 322 56 42 65 40 54 327909 327 132 130 88 132 96 327927 345 125120 114 120 108 327928 346 45 52 77 39 57 327933 351 127 132 82 127 100327937 355 81 77 76 63 92 327951 369 76 100 91 81 84 327953 371 94 94 92112 90 327956 374 80 90 102 69 91 327960 378 47 52 52 34 76 328093 39559 89 97 73 99 328112 414 92 89 73 97 79 328114 416 110 134 123 116 106328132 434 120 89 81 67 94 328340 449 76 130 85 112 110 328362 471 73 8359 80 78 328400 509 60 40 34 18 67 328417 526 83 98 87 68 94 328434 54391 96 85 83 79 328651 560 93 109 84 78 106 328677 586 34 68 61 44 89328685 594 50 100 73 69 91 328691 600 130 156 166 144 105 328759 668 87105 108 66 95

For these data, values for triglyceride accumulation above 100 areconsidered to indicate that the compound has the ability to stimulatetriglyceride accumulation, whereas values at or below 100 indicate thatthe compound inhibits triglyceride accumulation. With respect to leptinsecretion, values above 100 are considered to indicate that the compoundhas the ability to stimulate secretion of the leptin hormone, and valuesat or below 100 indicate that the compound has the ability to inhibitsecretion of leptin. With respect to the four adipocyte differentiationhallmark genes, values above 100 are considered to indicate induction ofcell differentiation, whereas values at or below 100 indicate that thecompound inhibits differentiation.

Several compounds were found to have remarkable effects. For example,the oligomeric compounds ISIS Number 327904 (SEQ ID NO: 322), targetedto mir-181a-1, ISIS Number 327928 (SEQ ID NO: 346), targeted to mir-29a,ISIS Number 327960 (SEQ ID NO: 378), targeted to mir-215, ISIS Number328400 (SEQ ID NO: 509), targeted to mir-196-2, and ISIS Number 328677(SEQ ID NO: 586), targeted to hypothetical miRNA-120 were shown toreduce the expression levels of all five markers of adipocytedifferentiation, indicating that these oligomeric compounds have theability to block adipocyte differentiation. Therefore, these oligomericcompounds may be useful as therapeutic agents with applications in thetreatment, attenuation or prevention of obesity, hyperlipidemia,atherosclerosis, atherogenesis, diabetes, hypertension, or othermetabolic diseases as well as in the maintenance of the pluripotentphenotype of stem or precursor cells.

The oligomeric compounds ISIS Number 328691 (SEQ ID NO: 600) targeted tomir-145, ISIS Number 328114 (SEQ ID NO: 416) targeted to hypotheticalmiRNA-138, and ISIS Number 327927 (SEQ ID NO: 345) targeted to mir-15bare examples of compounds which exhibit an increase in all five markersof adipocyte differentiation. Additionally, the oligomeric compound ISISNumber 327909 (SEQ ID NO: 327) targeted to mir-196-2 exhibited anincrease in three of the five markers of adipocyte differentiation.Thus, these oligomeric compounds may be useful as pharmaceutical agentsin the treatment of diseases in which the induction of adipocytedifferentiation is desirable, such as anorexia, or for conditions orinjuries in which the induction of cellular differentiation isdesirable, such as Alzheimers disease or central nervous system injury,in which regeneration of neural tissue would be beneficial. Furthermore,these oligomeric compounds may be useful in the treatment, attenuationor prevention of diseases in which it is desirable to induce cellulardifferentiation and/or quiescence, for example in the treatment ofhyperproliferative disorders such as cancer.

Example 32 Effects of Oligomeric Compounds on Endothelial Tube FormationAssay

Angiogenesis is the growth of new blood vessels (veins and arteries) byendothelial cells. This process is important in the development of anumber of human diseases, and is believed to be particularly importantin regulating the growth of solid tumors. Without new vessel formationit is believed that tumors will not grow beyond a few millimeters insize. In addition to their use as anti-cancer agents, inhibitors ofangiogenesis have potential for the treatment of diabetic retinopathy,cardiovascular disease, rheumatoid arthritis and psoriasis (Carmelietand Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell.Cardiol, 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465;Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt,Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol.Histopathol, 1999, 14, 1287-1294).

Endothelial Tube Formation Assay as a Measure of Angiogenesis:

Angiogenesis is stimulated by numerous factors that promote interactionof endothelial cells with each other and with extracellular matrixmolecules, resulting in the formation of capillary tubes. Thismorphogenic process is necessary for the delivery of oxygen to nearbytissues and plays an essential role in embryonic development, woundhealing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407,249-257). Moreover, this process can be reproduced in a tissue cultureassay that evaluates the formation of tube-like structures byendothelial cells. There are several different variations of the assaythat use different matrices, such as collagen I (Kanayasu et al.,Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem.,1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998,238, 324-334), as growth substrates for the cells. In this assay, humanumbilical vein endothelial cells (HuVECs) are plated on a matrix derivedfrom the Engelbreth-Holm-Swarm mouse tumor, which is very similar toMatrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri andPratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HuVECs formtube-like structures when grown on this substrate. Loss of tubeformation in vitro has been correlated with the inhibition ofangiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257;Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the useof in vitro tube formation as an endpoint for angiogenesis.

In one embodiment, primary human umbilical vein endothelial cells(HuVECs) were used to measure the effects of oligomeric compoundstargeted to miRNAs on tube formation activity. HuVECs were routinelycultured in EBM (Clonetics Corporation, Walkersville, Md.) supplementedwith SingleQuots supplements (Clonetics Corporation, Walkersville, Md.).Cells were routinely passaged by trypsinization and dilution when theyreached 90% confluence and were maintained for up to 15 passages. HuVECsare plated at 3000 cells/well in 96-well plates. One day later, cellsare transfected with oligomeric compounds. The tube formation assay isperformed using an in vitro Angiogenesis Assay Kit (ChemiconInternational, Temecula, Calif.).

A scrambled control compound, ISIS 29848 NNNNNNNNNNNNNNNNNNNN; where Nis A, T, C or G; herein incorporated as SEQ ID NO: 737) served as anegative control. ISIS 196103 (AGCCCATTGCTGGACATGCA; incorporated hereinas SEQ ID NO: 940) targets integrin beta 3 and was used as a positivecontrol to inhibit endothelial tube formation. ISIS 29248 and ISIS196103 are chimeric 5-10-52′-MOE gapmer oligonucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotides. All cytidine residues are5-methylcytidines. ISIS 342672 (SEQ ID NO: 789) contains 13 mismatcheswith respect to the mature mir-143 miRNA, and was also used as anegative control. ISIS 342672 is a uniform 2′-MOE phosphorothioateoligomeric compound 22 nucleotides in length. All cytidine residues are5-methylcytidines.

Oligomeric compound was mixed with LIPOFECTIN™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) in OPTI-MEM™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve a final concentration of 75nM of oligomeric compound and 2.25 μg/mL LIPOFECTIN™. Before adding tocells, the oligomeric compound, LIPOFECTIN™ and OPTI-MEM™ were mixedthoroughly and incubated for 0.5 hrs. Untreated control cells receivedLIPOFECTIN™ only. The medium was removed from the plates and the plateswere tapped on sterile gauze. Each well was washed in 150 μL ofphosphate-buffered saline. The wash buffer in each well was replacedwith 100 μL of the oligomeric compound/OPTI-MEM™/LIPOFECTIN™ cocktail.Compounds targeted to miRNAs were tested in triplicate, and ISIS 29848was tested in up to six replicates. The plates were incubated for 4hours at 37° C., after which the medium was removed and the plate wastapped on sterile gauze. 100 μL of full growth medium was added to eachwell. Fifty hours after transfection, cells are transferred to 96-wellplates coated with ECMa-trix™ (Chemicon Inter-national). Under theseconditions, untreated HuVECs form tube-like structures. After anovernight incubation at 37° C., treated and untreated cells areinspected by light microscopy. Individual wells are assigned discretescores from 1 to 5 depending on the extent of tube formation. A score of1 refers to a well with no tube formation while a score of 5 is given towells where all cells are forming an extensive tubular network. Resultsare expressed as a percentage of the level of the tube formationobserved in cultures not treated with oligonucleotide, and are shown inTables 56-59.

TABLE 56 Effect of compounds targeting miRNAs on Tube Formation Activityin HuVECs SEQ ID % Activity Relative ISIS NO: NO: Pri-miRNA to UTC UTCN/A N/A 100 196103 940 Integrin beta 3 35.7 positive control 342672 789N/A 46.4 negative control 327873 291 mir-140 100.0 327875 293 mir-3471.4 327876 294 mir-29b-1 50.0 327877 295 mir-16-3 78.6 327878 296mir-203 57.1 327879 297 mir-7-1 71.4 327880 298 mir-10b 57.1 327881 299mir-128a 50.0 327882 300 mir-153-1 107.1 327883 301 mir-27b 92.9 327884302 mir-96 78.6 327885 303 mir-17as/mir-91 50.0 327886 304mir-123/mir-126as 42.9 327887 305 mir-132 57.1 327888 306 mir-108-1100.0 327889 307 mir-23b 50.0 327890 308 let-7i 92.9 327891 309 mir-21250.0 327892 310 mir-131-2/mir-9 57.1 327893 311 let-7b 100.0 327894 312mir-1d 100.0 327895 313 mir-122a 100.0 327896 314 mir-22 64.3 327898 316mir-142 100.0

From these data, it was observed that ISIS Number 327886 targeted tomir-123/mir126 as suppressed tube formation, indicating that thiscompound may be useful as an angiogenesis inhibitor and/or anti-tumoragent, with potential therapeutic applications in the treatment ofdiabetic retinopathy, cardiovascular disease, rheumatoid arthritis,psoriasis, as well as cancer.

TABLE 57 Effect of compounds targeting miRNAs on Tube Formation Activityin HuVECs SEQ ID % Activity ISIS NO: NO: Pri-miRNA Relative to UTC UTCN/A N/A 100 196103 940 Integrin beta 3 24.1 positive control 342672 789N/A 58.6 negative control 327899 317 mir-183 34.5 327900 318 mir-21455.2 327901 319 mir-143 48.3 327902 320 mir-192-1 41.4 327903 321let-7a-3 103.5 327904 322 mir-181a 89.7 327905 323 mir-205 48.3 327906324 mir-103-1 69.0 327907 325 mir-26a 62.1 327908 326 mir-33a 103.5327909 327 mir-196-2 96.6 327910 328 mir-107 55.2 327911 329 mir-10675.9 327913 331 mir-29c 69.0 327914 332 mir-130a 82.8 327915 333mir-218-1 69.0 327916 334 mir-124a-2 96.6 327917 335 mir-21 82.8 327918336 mir-144 96.6 327919 337 mir-221 103.5 327920 338 mir-222 41.4 327921339 mir-30d 96.6 327922 340 mir-19b-2 89.7 327923 341 mir-128b 48.3

From these data, it was observed that ISIS Number 327899 targeted tomir-183, ISIS Number 327902 targeted to mir-192-1, and ISIS Number327920 targeted to mir-222 suppressed tube formation, indicating thatthese compounds may be useful as an angiogenesis inhibitors and/oranti-tumor agents, with potential therapeutic applications in thetreatment of diabetic retinopathy, cardiovascular disease, rheumatoidarthritis, psoriasis, as well as cancer.

TABLE 58 Effect of compounds targeting miRNAs on Tube Formation Activityin HuVECs SEQ ID % Activity Relative ISIS NO: NO: Pri-miRNA to UTC UTCN/A N/A 100 196103 940 Integrin beta 3 29.6 positive control 342672 789N/A 55.6 negative control 327924 342 mir-129-2 88.9 327925 343 mir-133b44.4 327926 344 let-7d 96.3 327927 345 mir-15b 59.3 327928 346 mir-29a-137.0 327929 347 mir-199b 51.9 327930 348 let-7e 88.9 327931 349 let-7c103.7 327932 350 mir-204 51.9 327933 351 mir-145 59.3 327934 352mir-213/mir-181a 51.9 327935 353 mir-20 74.1 327936 354 mir-133a-1 51.9327937 355 mir-138-2 88.9 327938 356 mir-98 96.3 327939 357 mir-125b-166.7 327940 358 mir-199a-2 59.3 327941 359 mir-181b 74.1 327942 360mir-141 74.1 327943 361 mir-18 81.5 327944 362 mir-220 37.0 327945 363mir-24-2 59.3 327946 364 mir-211 51.9 327947 365 mir-101-3 81.5

From these data, it was observed that ISIS Number 327925 targeted tomir-133b, ISIS Number 327928 targeted to mir-29a-1, and ISIS Number327944 targeted to mir-220 suppressed tube formation, indicating thatthese compounds may be useful as an angiogenesis inhibitors and/oranti-tumor agents, with potential therapeutic applications in thetreatment of diabetic retinopathy, cardiovascular disease, rheumatoidarthritis, psoriasis, as well as cancer.

TABLE 59 Effect of compounds targeting miRNAs on Tube Formation Activityin HuVECs SEQ ID % Activity Relative ISIS Number NO: Pri-miRNA to UTCUTC N/A N/A 100 196103 940 Integrin beta 3 26.7 positive control 342672789 N/A 60.0 negative control 327874 292 mir-30a 46.7 327897 315mir-92-1 40.0 327901 319 mir-143 100.0 327948 366 mir-30b 33.3 327949367 mir-10a 66.7 327950 368 mir-19a 73.3 327951 369 mir-15a-1 73.3327952 370 mir-137 53.3 327953 371 mir-219 53.3 327954 372 mir-148b 53.3327955 373 mir-130b 46.7 327956 374 mir-216 46.7 327957 375 mir-100-166.7 327958 376 mir-187 40.0 327959 377 mir-210 40.0 327960 378 mir-21553.3 327961 379 mir-223 53.3 327962 380 mir-30c 53.3 327963 381 mir-26b93.3 327964 382 mir-152 86.7 327965 383 mir-135-1 100.0 327966 384mir-217 40.0 327967 385 let-7g 93.3 327968 386 mir-33b 93.3

From these data, it was observed that ISIS Number 327948 targeted tomir-30b, ISIS Number 327958 targeted to mir-187, ISIS Number 327959targeted to mir-210, and ISIS Number 327966 targeted to mir-217suppressed tube formation, indicating that these compounds may be usefulas an angiogenesis inhibitors and/or anti-tumor agents, with potentialtherapeutic applications in the treatment of diabetic retinopathy,cardiovascular disease, rheumatoid arthritis, psoriasis, as well ascancer.

Example 33 Effect of Oligomeric Compounds on miRNA Target ProteinExpression

Several mRNA transcripts have been predicted to be regulated by miRNAs(Lewis et al., Cell, 2003, 115, 787-798). For example, the mRNAs encodedby six genes, 1) inwardly rectifying potassium channel Kir2.2 (GenBankAccession AB074970, SEQ ID NO: 872); 2) synaptotagmin III (GenBankAccession BC028379, SEQ ID NO: 873); 3) mitogen-activated protein kinase7/extracellular signal-regulated kinase 5 (ERK5) (GenBank AccessionNM_(—)139032.1, SEQ ID NO: 861); 4) protein phosphatase 2 (formerly 2A),catalytic subunit, beta isoform (PPP2CB) (GenBank AccessionNM_(—)004156.1, SEQ ID NO: 814); 5) glyoxalase I (GenBank AccessionNM_(—)006708.1, SEQ ID NO: 821); and 6) LIM domain only 4 (LMO4)(GenBank Accession NM_(—)006769.2, SEQ ID NO: 865), are believed to havemir-143 binding sites within their 3′-UTRs. The latter three genesencode mRNAs that were identified as potential targets of mir-143 by theRACE-PCR experiments described, supra. Thus, the mir-143 miRNA ispredicted to regulate some or all of these genes.

When miRNAs have effects on the expression of downstream genes orproteins encoded by these genes, it is advantageous to measure theprotein levels of those gene products, and to do this, western blot(immunoblot) analysis is employed. Immunoblot analysis is carried outusing standard methods. Briefly, preadipocytes and differentiatingadipocytes were cultured as described previously, and differentiatingadipocytes are sampled at several timepoints after stimulation ofdifferentiation. Cells were treated with 250 nM oligomeric compounds andharvested 16-20 h after oligomeric compound treatment. Cells werewashed, lysed in RIPA buffer with protease inhibitor cocktail (RocheDiagnostics Corporation, Indianapolis, Ind.), suspended in Laemmlibuffer (20 ul/well), boiled for 5 minutes and loaded onto either an 8%SDS-PAGE or a 4-20% gradient SDS-PAGE gel. Gels are run forapproximately 1.5 hours at 150 V, and transferred to PVDF membrane forwestern blotting. Appropriate primary antibody directed to a target isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Because expression levelsof the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein remainconstant, an antibody recognizing the GAPDH protein (Abcam, Cambridge,Mass.) can be used in a re-probing of the membrane to verify equalprotein loading. It is also understood that antisense oligomericcompounds specifically targeting and known to inhibit the expression ofthe mRNA and protein endproducts of the gene of interest can be used ascontrols in these experiments. Bands are visualized and quantitatedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.) or theChemiDoc™ system (Bio-Rad Laboratories, Inc., Hercules, Calif.). Thus,the effects of treatment of many cell types (including, but not limitedto, preadipocytes, differentiating adipocytes, HeLa, T-24 and A549cells) with oligomeric compounds of the present invention on the levelsof gene expression products can be assessed. It is understood that oneof ordinary skill in the art can use immunoblot analysis to examine theexpression of any protein predicted to be the downstream expressionproduct of a target of a miRNA. Similarly, using methods describedabove, real-time RT-PCR methods can also be used to examine the mRNAexpression levels of any of these predicted targets of the mir-143miRNA. More specifically, immunoblot analysis and/or real-time RT-PCRmethods can be used to examine the effects of treatment with oligomericcompounds on the protein or mRNA levels, respectively, produced by theKir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LM04 genesin a variety of cell types.

In one embodiment of the invention, immunoblot analysis was used toassess the effects of the oligomeric compound, ISIS Number 327901 (SEQID NO: 319) targeting mir-143, on expression levels of the PPP2CBprotein in differentiating adipocytes. It was observed that, upontreatment with ISIS 327901, PPP2CB protein levels were higher indifferentiating adipocytes both 7- and 10-days post-differentiation thanin pre-adipocytes or in untreated differentiating adipocytes from thesame timepoints. Thus, mir-143 appears to negatively regulate theexpression of the PPP2CB gene, presumably by inhibiting translation ofthe PPP2CB mRNA into protein, and upon treatment with the oligomericcompound ISIS 327901, this inhibition of PPP2CB protein expression wasrelieved.

In one embodiment of the invention, immunoblot analysis was used toassess the effects of the oligomeric compound, ISIS Number 327901 (SEQID NO: 319) targeting mir-143, on expression levels of the ERK5 proteinin differentiating adipocytes. It was observed that, upon treatment ofcells with ISIS 327901, ERK5 protein levels were approximately2-2.5-fold higher in differentiating adipocytes both 7- and 10-dayspost-differentiation than in pre-adipocytes or in untreateddifferentiating adipocytes from the same timepoints. Thus, mir-143appears to negatively regulate the expression of the ERK5 genepresumably by inhibiting translation of the ERK5 mRNA into protein,either directly (by mir-143 binding an ERK5 cis-regulatory sequence) orindirectly (by mir-143 regulating another target gene that regulatesERK5); upon treatment with the oligomeric compound ISIS 327901, thismir-143-dependent inhibition of ERK5 expression was relieved. It isknown that ERK5 promotes cell growth and proliferation in response totyrosine kinase signaling. In light of the involvement of mir-143 inadipocyte differentiation disclosed in several examples in the presentinvention, as well as the role of mir-143 in regulating ERK5, it ispredicted that ERK5 and mir-143 are together involved regulating thebalance between cellular proliferation and differentiation.

It is understood that the oligomeric compounds of the present invention,including miRNA mimics, can also be tested for their effects on theexpression of the protein endproducts of targets of miRNAs. For example,an oligomeric compound such as a mir-143 mimic can be used to treatdifferentiating adipocytes, and is predicted to result in a reduction ofKir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LM04protein expression levels.

The phosphatase and tensin homolog (mutated in multiple advancedcancers 1) (PTEN) tumor suppressor mRNA (GenBank Accession NM_(—)000314,incorporated herein as SEQ ID NO: 941) has been predicted to be apotential target of the mir-19a miRNA (Lewis et al., Cell, 2003, 115,787-798). Oligomeric compounds that target or mimic the mir-19a miRNA ormir-19a pri-miRNA can be used to treat cells and, using the methodsdescribed above, the effects of these oligomeric compounds on theexpression of the PTEN protein and mRNA levels can be assessed. It ispredicted that the mir-19a miRNA, or an oligomeric compound acting as amir-19a mimic, would inhibit expression of the PTEN tumor suppressormRNA and protein, and that treatment with oligomeric compounds targetingmir-19a would reverse this inhibition. It is also understood that otherantisense oligomeric compounds specifically targeting and known toinhibit the expression of the mRNA and protein endproducts of the geneinterest can be used as controls in these experiments.

Example 34 Additional Oligomeric Compounds Targeting miRNAs

In accordance with the present invention, oligomeric compounds weredesigned and synthesized to target or mimic one or more miRNA genes orgene products. Pri-miRNAs, pre-miRNAs and mature miRNAs represent targetnucleic acids to which the oligomeric compounds of the present inventionwere designed and synthesized. Oligomeric compounds of the presentinvention can also be designed and synthesized to mimic the pri-miRNA,pre-miRNA or mature miRNA structure while incorporating certain chemicalmodifications that alter one or more properties of the mimic, thuscreating a construct with superior properties as compared to theendogenous precursor or mature miRNA.

In accordance with the present invention, oligomeric compounds weredesigned to target or mimic one or more human, mouse, rat, or Drosophilapri-miRNAs, pre-miRNAs or mature miRNAs.

A list of human pri-miRNAs and the mature miRNAs predicted to derivefrom them is shown in Table 60. “Pri-miRNA name” indicates the gene namefor each of the pri-miRNAs. Also given in table 60 are the name andsequence of the mature miRNA derived from the pri-miRNA. Mature miRNAsequences from pri-miRNA precursors have been proposed by severalgroups; consequently, for a given pri-miRNA sequence, several miRNAs maybe disclosed and given unique names, and thus a given pri-miRNA sequencemay occur repeatedly in the table. The sequences are written in the 5′to 3′ direction and are represented in the DNA form. It is understoodthat a person having ordinary skill in the art would be able to convertthe sequence of the targets to their RNA form by simply replacing thethymidine (T) with uracil (U) in the sequence.

TABLE 60 Human pri-miRNA sequences and the corresponding mature miRNAsSEQ ID SEQ ID Pri-miRNA name NO Mature miRNA name Mature miRNA sequenceNO mir-27b 17 mir-27b TTCACAGTGGCTAAGTTCTG 202 mir-27b 17 miR-27*(Michael TTCACAGTGGCTAAGTTCTGC 1059 et al) mir-23b 23 mir-23bATCACATTGCCAGGGATTACCAC 208 glutamate 36 hypotheticalTGTTATAGTATTCCACCTACC 1060 receptor, miRNA-033 ionotrophic, AMPA3/hypothetical miRNA-033 LOC 114614 74 hypotheticalTGCTAATCGTGATAGGGGTTT 1061 containing miR- miRNA-071 155/hypotheticalmiRNA-071 LOC 114614 74 mir-155 (RFAM) TTAATGCTAATCGTGATAGGGG 1062containing miR- 155/hypothetical miRNA-071 collagen, type I, 147hypothetical AGACATGTTCAGCTTTGTGGA 1063 alpha 1/ miRNA-144 hypotheticalmiRNA-144 sterol regulatory 168 mir-33b GTGCATTGCTGTTGCATTG 286element-binding protein-1/mir-33b tight junction 186 hypotheticalAGCCTGTGGAGCTGCGCTTAC 1064 protein 1 (zona miRNA-183 occludens 1)/hypothetical miRNA-183 mir-140 4 mir-140 AGTGGTTTTACCCTATGGTAG 192mir-140 4 miR-140-as TACCACAGGGTAGAACCACGGA 1065 mir-140 4 mir-239*(Kosik) TACCACAGGGTAGAACCACGGACA 1066 mir-34 6 mir-34TGGCAGTGTCTTAGCTGGTTGT 194 mir-34 6 miR-172 (RFAM-M. mu.)TGGCAGTGTCTTAGCTGGTTGTT 1067 mir-203 10 mir-203 GTGAAATGTTTAGGACCACTAG197 mir-203 10 miR-203 (RFAM-M. mu.) TGAAATGTTTAGGACCACTAG 1068 mir-20310 miR-203 (Tuschl) TGAAATGTTTAGGACCACTAGA 1069 mir-7_1/mir-7_1* 11mir-7_1*_Ruvkun CAACAAATCACAGTCTGCCATA 1070 mir-7_1/mir-7_1* 11 mir-7TGGAAGACTAGTGATTTTGTT 198 mir-10b 12 miR-10b (Tuschl)CCCTGTAGAACCGAATTTGTGT 1071 mir-10b 12 mir-10b TACCCTGTAGAACCGAATTTGT199 mir-10b 12 miR-10b (Michael TACCCTGTAGAACCGAATTTGTG 1072 et al)mir-128a 13 mir-128 (Kosik) TCACAGTGAACCGGTCTCTTT 1073 mir-128a 13mir-128a TCACAGTGAACCGGTCTCTTTT 200 mir-153_1 14 mir-153TTGCATAGTCACAAAAGTGA 201 mir-153_2 15 mir-153 TTGCATAGTCACAAAAGTGA 201hypothetical miR- 16 hypothetical TATCAAACATATTCCTACAGT 1074 13/miR-190miRNA-013 hypothetical miR- 16 miR-190 TGATATGTTTGATATATTAGGT 107513/miR-190 mir-123/mir-126 20 mir-123/mir-126as CATTATTACTTTTGGTACGCG205 mir-123/mir-126 20 mir-126 TCGTACCGTGAGTAATAATGC 1076 mir-132 21miR-132 (RFAM- TAACAGTCTACAGCCATGGTCG 1077 Human) mir-132 21 mir-132TAACAGTCTACAGCCATGGTCGC 206 mir-108_1 22 mir-108 ATAAGGATTTTTAGGGGCATT207 let-7i 24 let-7i TGAGGTAGTAGTTTGTGCT 209 let-7i 24 let-7i_RuvkunTGAGGTAGTAGTTTGTGCTGTT 1078 mir-212 25 mir-212 TAACAGTCTCCAGTCACGGCC 210hypothetical miRNA 26 hypothetical TGGGCAAGAGGACTTTTTAAT 1079 023miRNA-023 mir-131_2/mir-9 27 mir-131 TAAAGCTAGATAACCGAAAGT 211mir-131_2/mir-9 27 mir-131_Ruvkun TAAAGCTAGATAACCGAAAGTA 1080mir-131_2/mir-9 27 miR-9 TCTTTGGTTATCTAGCTGTATGA 1081 let-7b 28 let-7bTGAGGTAGTAGGTTGTGTGGTT 212 let-7b 28 let-7b_RuvkunTGAGGTAGTAGGTTGTGTGGTTT 1082 mir-1d_1 29 miR-1 (RFAM)TGGAATGTAAAGAAGTATGTA 1083 mir-1d_1 29 mir-1d TGGAATGTAAAGAAGTATGTAT 213mir-122a 30 miR-122a, b TGGAGTGTGACAATGGTGTTTG 1084 (Tuschl) mir-122a 30mir-122a TGGAGTGTGACAATGGTGTTTGT 214 mir-22 31 mir-22AAGCTGCCAGTTGAAGAACTGT 215 hypothetical miRNA 33 hypotheticalTGACATCACATATACGGCAGC 1085 30 miRNA-030 mir-142 34 mir-142CATAAAGTAGAAAGCACTAC 217 mir-142 34 miR-142-as TGTAGTGTTTCCTACTTTATGG1086 mir-142 34 miR-142as TGTAGTGTTTCCTACTTTATGGA 1087 (Michael et al)mir-183 35 mir-183 TATGGCACTGGTAGAATTCACTG 218 mir-214 37 mir-214ACAGCAGGCACAGACAGGCAG 219 mir-143 38 miR-143 (MichaelTGAGATGAAGCACTGTAGCTC 1088 et al) mir-143 38 mir-143TGAGATGAAGCACTGTAGCTCA 220 mir-192_1 39 miR-192 (Tuschl)CTGACCTATGAATTGACA 1089 mir-192_1 39 mir-192 CTGACCTATGAATTGACAGCC 221mir-192_1 39 miR-192 (Michael TGACCTATGAATTGACAGCCAG 1090 et al)hypothetical miRNA 42 hypothetical TAAGACTTGCAGTGATGTTTA 1091 039miRNA-039 hypothetical miRNA 43 hypothetical TGTCAACAAAACTGCTTACAA 1092040 miRNA-040 hypothetical miRNA 44 hypothetical TACCAGTTGTTTTCTCTGTGA1093 041 miRNA-041 let-7a_3 45 let-7a TGAGGTAGTAGGTTGTATAGTT 222hypothetical miRNA 46 hypothetical TGACAGGAAATCTTTGAGAGG 1094 043miRNA-043 hypothetical miRNA 47 hypothetical TTCCACTCTGTTTATCTGACA 1095044 miRNA-044 mir-181a_1 48 mir-178 (Kosik) AACATTCAACGCTGTCGGTGAG 1096mir-181a_1 48 mir-181a AACATTCAACGCTGTCGGTGAGT 223 let-7a_1 49 let-7aTGAGGTAGTAGGTTGTATAGTT 222 mir-205 50 mir-205 TCCTTCATTCCACCGGAGTCTG 224mir-33a 53 mir-33a GTGCATTGTAGTTGCATTG 227 mir-196_2 54 miR-196 (Tuschl)TAGGTAGTTTCATGTTGTTGG 1097 mir-196_2 54 mir-196 TAGGTAGTTTCATGTTGTTGGG228 let-7f_1 57 let-7f (Michael TGAGGTAGTAGATTGTATAGT 1098 et al)let-7f_1 57 let-7f TGAGGTAGTAGATTGTATAGTT 231 hypothetical miRNA 58hypothetical TTGCATGCCCTATTGATTCTC 1099 055 miRNA-055 mir-29c 59 mir-29cCTAGCACCATTTGAAATCGGTT 232 mir-29c 59 miR-29c (Tuschl)TAGCACCATTTGAAATCGGTTA 1100 mir-130a 60 mir-130a CAGTGCAATGTTAAAAGGGC233 mir-130a 60 mir-130 (Kosik) CAGTGCAATGTTAAAAGGGCAT 1101 hypotheticalmiRNA 61 hypothetical TGTCAGATGCTTAATGTTCTT 1102 058 miRNA-058 mir-218_162 mir-218 TTGTGCTTGATCTAACCATGT 234 mir-218_1 62 mir-253* (Kosik)TTGTGCTTGATCTAACCATGTG 1103 mir-124a_2 63 mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA 1104 mir-124a_2 63 mir-124a TTAAGGCACGCGGTGAATGCCA235 mir-124a_2 63 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 mir-14466 mir-144 TACAGTATAGATGATGTACTAG 237 mir-221 67 mir-221 (RFAM-AGCTACATTGTCTGCTGGGTTT 1106 mmu) mir-221 67 mir-221AGCTACATTGTCTGCTGGGTTTC 238 mir-222 68 mir-222 (RFAM-AGCTACATCTGGCTACTGGGTCT 1107 mmu) mir-222 68 mir-222AGCTACATCTGGCTACTGGGTCTC 239 mir-30d 69 mir-30d TGTAAACATCCCCGACTGGAAG240 mir-30d 69 mir-30d_Ruvkun TGTAAACATCCCCGACTGGAAGCT 1108 mir-128b 71mir-128 (Kosik) TCACAGTGAACCGGTCTCTTT 1073 mir-128b 71 mir-128bTCACAGTGAACCGGTCTCTTTC 242 mir-219_2 72 mir-219 TGATTGTCCAAACGCAATTCT271 hypothetical miRNA 73 hypothetical TCACATTTGCCTGCAGAGATT 1109 070miRNA-070 mir-129_2 76 mir-129as/mir- AAGCCCTTACCCCAAAAAGCAT 1110 258*(Kosik) mir-129_2 76 mir-129 CTTTTTGCGGTCTGGGCTTGC 243 mir-129_2 76miR-129b (RFAM- CTTTTTGCGGTCTGGGCTTGCT 1111 Human) mir-133b 77 mir-133bTTGGTCCCCTTCAACCAGCTA 244 hypothetical miRNA 78 hypotheticalTGGTTAAAATATTAATGGGGC 1112 075 miRNA-075 let-7d 79 let-7dAGAGGTAGTAGGTTGCATAGT 245 let-7d 79 let-7d_Ruvkun AGAGGTAGTAGGTTGCATAGTT1113 let-7d 79 let-7d* (RFAM-M. mu.) CTATACGACCTGCTGCCTTTCT 1114 mir-15b80 miR-15b (Michael TAGCAGCACATCATGGTTTAC 1115 et al) mir-15b 80 mir-15bTAGCAGCACATCATGGTTTACA 246 mir-29a 81 mir-29a CTAGCACCATCTGAAATCGGTT 247mir-29a 81 mir-29a_Ruvkun TAGCACCATCTGAAATCGGTTA 1116 hypothetical miRNA82 hypothetical TGATATGTTTGATATTGGG 1117 079 miRNA-079 mir-199b 83mir-199b (human) CCCAGTGTTTAGACTATCTGTTC 248 mir-199b 83 miR-199-asTACAGTAGTCTGCACATTGGTT 1118 mir-129_1 84 mir-129 CTTTTTGCGGTCTGGGCTTGC243 mir-129_1 84 miR-129b (RFAM- CTTTTTGCGGTCTGGGCTTGCT 1111 Human)let-7e 85 let-7e TGAGGTAGGAGGTTGTATAGT 249 hypothetical miRNA 86hypothetical TTACATGGGGAAGCTATCATA 1119 083 miRNA-083 let-7c_1 87 let-7cTGAGGTAGTAGGTTGTATGGTT 250 let-7c_1 87 let-7c_RuvkunTGAGGTAGTAGGTTGTATGGTTT 1120 mir-204 88 mir-204 TTCCCTTTGTCATCCTATGCCT251 mir-204 88 miR-204 (Tuschl) TTCCCTTTGTCATCCTATGCCTG 1121 mir-145 89miR-145 (Michael GTCCAGTTTTCCCAGGAATCC 1122 et al) mir-145 89 mir-145GTCCAGTTTTCCCAGGAATCCCTT 252 mir-124a_1 90 mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA 1104 mir-124a_1 90 mir-124a TTAAGGCACGCGGTGAATGCCA235 mir-124a_1 90 mir-124a_Ruvkun TTAAGGCACGCGGTGAATGCCAA 1105 DiGeorgesyndrome 91 hypothetical TGTGATTTCCAATAATTGAGG 1123 critical regionmiRNA-088 gene 8/ hypothetical miRNA-088 mir-213/mir- 92 mir-178 (Kosik)AACATTCAACGCTGTCGGTGAG 1096 181a_2 mir-213/mir- 92 mir-181aAACATTCAACGCTGTCGGTGAGT 223 181a_2 mir-213/mir- 92 mir-213ACCATCGACCGTTGATTGTACC 253 181a_2 hypothetical miRNA 93 hypotheticalTAGGCCAAATGGCGCATCAAT 1124 090 miRNA-090 mir-20 94 miR-20* (human)ACTGCATTATGAGCACTTAAA 1125 mir-20 94 miR-20 (RFAM-TAAAGTGCTTATAGTGCAGGTA 1126 Human) mir-20 94 mir-20TAAAGTGCTTATAGTGCAGGTAG 254 mir-133a_1 95 mir-133aTTGGTCCCCTTCAACCAGCTGT 255 mir-138_2 96 mir-138 AGCTGGTGTTGTGAATC 256mir-138_2 96 mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127 mir-196_1 98miR-196 (Tuschl) TAGGTAGTTTCATGTTGTTGG 1097 mir-196_1 98 mir-196TAGGTAGTTTCATGTTGTTGGG 228 mir-125b_1 99 mir-125b TCCCTGAGACCCTAACTTGTGA258 mir-199a_2 100 miR-199-s CCCAGTGTTCAGACTACCTGTT 1128 mir-199a_2 100mir-199a CCCAGTGTTCAGACTACCTGTTC 259 mir-199a_2 100 miR-199-asTACAGTAGTCTGCACATTGGTT 1118 hypothetical miRNA 102 hypotheticalAGGCAGATAGAGAAGTCACAG 1272 099 miRNA-099 mir-181b_1 103 mir-181bAACATTCATTGCTGTCGGTGGGTT 260 hypothetical miRNA 104 hypotheticalTGACAGTCAATTAACAAGTTT 1130 101 miRNA-101 mir-141 105 mir-141AACACTGTCTGGTAAAGATGG 261 mir-131_1/mir-9 106 mir-131TAAAGCTAGATAACCGAAAGT 211 mir-131_1/mir-9 106 mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA 1080 mir-131_1/mir-9 106 miR-9TCTTTGGTTATCTAGCTGTATGA 1081 mir-133a_2 107 mir-133aTTGGTCCCCTTCAACCAGCTGT 255 hypothetical miRNA 108 miR-202 (human)AGAGGTATAGGGCATGGGAAAA 1131 105 hypothetical miRNA 108 hypotheticalTTCCTATGCATATACTTCTTT 1132 105 miRNA-105 hypothetical miRNA 110hypothetical TGACAGTTTATTGGCTTTATC 1133 107 miRNA-107 mir-1d_2 111 miR-1(RFAM) TGGAATGTAAAGAAGTATGTA 1083 mir-1d_2 111 mir-1dTGGAATGTAAAGAAGTATGTAT 213 mir-1d_2 111 miR-1d (Tuschl)TGGAATGTAAAGAAGTATGTATT 1134 mir-220 113 mir-220 CCACACCGTATCTGACACTTT263 hypothetical miRNA 114 hypothetical TTCCTCCTCCTCCGACTCGGA 1135 111miRNA-111 mir-7_3 115 mir-7 TGGAAGACTAGTGATTTTGTT 198 mir-218_2 116mir-218 TTGTGCTTGATCTAACCATGT 234 mir-218_2 116 mir-253* (Kosik)TTGTGCTTGATCTAACCATGTG 1103 mir-211 120 mir-211 (human)TTCCCTTTGTCATCCTTCGCCT 1136 mir-30b 122 mir-30b TGTAAACATCCTACACTCAGC266 mir-30b 122 mir-30b_Ruvkun TGTAAACATCCTACACTCAGCT 1137 hypotheticalmiRNA 123 hypothetical TTACAGCAATCCAGTAATGAT 1138 120 miRNA-120 mir-10a125 mir-10a (Tuschl) TACCCTGTAGATCCGAATTTGT 1139 mir-10a 125 mir-10aTACCCTGTAGATCCGAATTTGTG 267 let-7f_2 127 let-7f (MichaelTGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_2 127 let-7fTGAGGTAGTAGATTGTATAGTT 231 mir-108_2 129 mir-108 ATAAGGATTTTTAGGGGCATT207 mir-137 130 mir-137 TATTGCTTAAGAATACGCGTAG 270 mir-148b 132 mir-148bTCAGTGCATCACAGAACTTTGT 272 mir-130b 133 mir-130b CAGTGCAATGATGAAAGGGC273 mir-130b 133 mir-266* (Kosik) CAGTGCAATGATGAAAGGGCAT 1140 let-7a_4135 let-7a TGAGGTAGTAGGTTGTATAGTT 222 mir-216 136 mir-216TAATCTCAGCTGGCAACTGTG 274 hypothetical miRNA 140 hypotheticalTAAACTGGCTGATAATTTTTG 1141 137 miRNA-137 hypothetical miRNA 141hypothetical TGCAAGTATGAAAATGAGATT 1142 138 miRNA-138 mir-124a_3 143mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_3 143 mir-124aTTAAGGCACGCGGTGAATGCCA 235 mir-124a_3 143 mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA 1105 mir-7_2 144 mir-7 TGGAAGACTAGTGATTTTGTT 198hypothetical miRNA 145 hypothetical TGACGCTGCTCCCCACCTTCT 1143 142miRNA-142 hypothetical miRNA 146 hypothetical TGCAATTTGCTTGCAATTTTG 1144143 miRNA-143 mir-210 148 mir-210 CTGTGCGTGTGACAGCGGCTG 277 mir-215 149mir-215 ATGACCTATGAATTGACAGAC 278 mir-223 150 mir-223TGTCAGTTTGTCAAATACCCC 279 mir-131_3/mir-9 151 mir-131TAAAGCTAGATAACCGAAAGT 211 mir-131_3/mir-9 151 mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA 1080 mir-131_3/mir-9 151 miR-9TCTTTGGTTATCTAGCTGTATGA 1081 mir-199a_1 152 miR-199-sCCCAGTGTTCAGACTACCTGTT 1128 mir-199a_1 152 mir-199aCCCAGTGTTCAGACTACCTGTTC 259 mir-199a_1 152 miR-199-asTACAGTAGTCTGCACATTGGTT 1118 mir-30c_1 153 mir-30cTGTAAACATCCTACACTCTCAGC 280 mir-30c_1 153 mir-30c_RuvkunTGTAAACATCCTACACTCTCAGCT 1129 hypothetical miRNA 156 hypotheticalTGCAAGCAGATGCTGATAATA 1145 153 miRNA-153 hypothetical miRNA 157hypothetical TTAAAGTGGATGTGTGTTATT 1146 154 miRNA-154 mir-26b 158miR-26b (RFAM- TTCAAGTAATTCAGGATAGGT 1147 Human) mir-26b 158 mir-26bTTCAAGTAATTCAGGATAGGTT 281 hypothetical miRNA 159 hypotheticalTGCTTTCCCTCCTTCCTTCTT 1148 156 miRNA-156 mir-152 160 mir-152TCAGTGCATGACAGAACTTGG 282 mir-135_1 161 miR-135 (RFAM-TATGGCTTTTTATTCCTATGTGA 1149 Human) mir-135_1 161 mir-135TATGGCTTTTTATTCCTATGTGAT 283 non-coding RNA in 162 miR-135 (RFAM-TATGGCTTTTTATTCCTATGTGA 1149 rhabdomyosarcoma/ Human) mir-135_2non-coding RNA in 162 mir-135 TATGGCTTTTTATTCCTATGTGAT 283rhabdomyosarcoma/ mir-135_2 mir-217 163 mir-217 (human)TACTGCATCAGGAACTGATTGGAT 284 hypothetical miRNA 164 hypotheticalTGGCCATAAACTTGTAGTCAT 1150 161 miRNA-161 mir-15a 165 mir-15_RuvkunTAGCAGCACATAATGGTTTGT 1151 mir-15a 165 mir-15a TAGCAGCACATAATGGTTTGTG269 let-7g 166 let-7g TGAGGTAGTAGTTTGTACAGT 285 let-7g 166let-7gL_Ruvkun TGAGGTAGTAGTTTGTACAGTT 1152 hypothetical miRNA 167hypothetical TGCAAGGATTTTTATGTTTTG 1153 164 miRNA-164 hypothetical miRNA169 hypothetical TTCCAGTTGCAGCACCTGTAA 1154 166 miRNA-166 hypotheticalmiRNA 171 hypothetical AGCCAGGTGCCTTCACCTGCT 1155 168_1/similar tomiRNA-168 ribosomal protein L5 forkhead box 172 hypotheticalTGGCAGCTCTGGCATTTCATA 1156 P2/hypothetical miRNA-169 miRNA-169hypothetical miRNA 173 hypothetical TGATCTTGCTCTAACACTTGG 1157 170miRNA-170 glutamate 174 hypothetical TGACAAGTATGTTTTATCGTT 1158receptor, miRNA-171 ionotropic, AMPA 2/ hypothetical miRNA-171hypothetical miRNA 175 hypothetical TCCAACTGCAAGAAGTTACT 1159 172miRNA-172 hypothetical miRNA 176 hypothetical TAGTACGAGAAGAAGGAGGCT 1160173 miRNA-173 mir-182 177 miR-182* (RFAM- TGGTTCTAGACTTGCCAACTA 1161Human) mir-182 177 mir-182 TTTGGCAATGGTAGAACTCACA 287 hypothetical miRNA178 hypothetical TCTCCTTCAACCACCTGAGGT 1162 175 miRNA-175 hypotheticalmiRNA 179 hypothetical TAGGAGTTTGATATGACATAT 1163 176 miRNA-176hypothetical 180 hypothetical AGACAAACATGCTACTCTCAC 1164 miRNA-177_1miRNA-177 hypothetical miRNA 181 hypothetical TAGCCTATCTCCGAACCTTCA 1165178 miRNA-178 hypothetical miRNA 182 hypothetical TGAAAGGCACTTTGTCCAATT1166 179 miRNA-179 hypothetical miRNA 184 hypotheticalTCACCTGCTCTGGAAGTAGTT 1167 181 miRNA-181 mir-148a 185 mir-148aTCAGTGCACTACAGAACTTTGT 288 hypothetical miRNA 188 hypotheticalTGATGGCCAGCTGAGCAGCTC 1168 185 miRNA-185 hypothetical 189 hypotheticalAGACAAACATGCTACTCTCAC 1164 miRNA-177_2/ miRNA-177 hypothetical miRNA 186mir-181c 190 mir-181c AACATTCAACCTGTCGGTGAGT 290 hypothetical miRNA 191hypothetical TGGTGAGGGGAATGAAAAGTA 1169 188 miRNA-188 mir-100_1 945mir-100 AACCCGTAGATCCGAACTTGTG 275 mir-101_1 946 mir-101TACAGTACTGTGATAACTGA 265 mir-101_1 946 miR-101 (RFAM-TACAGTACTGTGATAACTGAAG 1170 Human) mir-101_3 947 mir-101TACAGTACTGTGATAACTGA 265 mir-101_3 947 miR-101 (RFAM-TACAGTACTGTGATAACTGAAG 1170 Human) mir-29b_2 948 miR-29b (RFAM-TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_2 948 miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT 1173 mir-29b_2 948 mir-29bTAGCACCATTTGAAATCAGTGTT 195 mir-29b_1 949 miR-29b (RFAM-TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_1 949 miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT 1173 mir-29b_1 949 mir-29bTAGCACCATTTGAAATCAGTGTT 195 mir-103_1 950 mir-103AGCAGCATTGTACAGGGCTATGA 225 mir-106 951 mir-106 (human)AAAAGTGCTTACAGTGCAGGTAGC 230 mir-107 952 mir-107 AGCAGCATTGTACAGGGCTATCA229 mir-16_1 953 mir-16 TAGCAGCACGTAAATATTGGCG 196 mir-16_1 953mir-16_Ruvkun TAGCAGCACGTAAATATTGGCGT 1176 mir-16_3 954 mir-16TAGCAGCACGTAAATATTGGCG 196 mir-16_3 954 mir-16_RuvkunTAGCAGCACGTAAATATTGGCGT 1176 mir-18 955 mir-18 TAAGGTGCATCTAGTGCAGATA262 mir-18 955 mir-18_Ruvkun TAAGGTGCATCTAGTGCAGATAG 1177 mir-19a 956mir-19a TGTGCAAATCTATGCAAAACTGA 268 mir-19b_1 957 mir-19b* (MichaelAGTTTTGCAGGTTTGCATCCAGC 1179 et al) mir-19b_1 957 mir-19bTGTGCAAATCCATGCAAAACTGA 241 mir-19b_2 958 mir-19bTGTGCAAATCCATGCAAAACTGA 241 mir-21 959 mir-21 TAGCTTATCAGACTGATGTTGA 236mir-23a 960 mir-23a ATCACATTGCCAGGGATTTCC 289 mir-24_2 961 mir-24TGGCTCAGTTCAGCAGGAACAG 264 mir-17/mir-91 962 mir-17 (human,ACTGCAGTGAAGGCACTTGT 1180 rat) mir-17/mir-91 962 mir-91_RuvkunCAAAGTGCTTACAGTGCAGGTAG 1181 mir-17/mir-91 962 mir-17as/mir-91CAAAGTGCTTACAGTGCAGGTAGT 204 mir-92_1 963 miR-92 (RFAM-M. mu.)TATTGCACTTGTCCCGGCCTG 1182 mir-92_1 963 mir-92 TATTGCACTTGTCCCGGCCTGT216 mir-96 964 mir-96 TTTGGCACTAGCACATTTTTGC 203 mir-96 964 miR-96(RFAM-M. mu.) TTTGGCACTAGCACATTTTTGCT 1183 mir-30a 965 mir-30aCTTTCAGTCGGATGTTTGCAGC 193 mir-30a 965 miR-30a-s TGTAAACATCCTCGACTGGAAGC1184 mir-98 966 mir-98 TGAGGTAGTAAGTTGTATTGTT 257 mir-104 967 miR-104TCAACATCAGTCTGATAAGCTA 335 (Mourelatos) (Mourelatos) mir-105 968 miR-105TCAAATGCTCAGACTCCTGT 1185 (Mourelatos) (Mourelatos) mir-27 969 miR-27TTCACAGTGGCTAAGTTCC 1186 (Mourelatos) (Mourelatos) mir-27 969 miR-27a(RFAM-M. mu.) TTCACAGTGGCTAAGTTCCGC 1187 (Mourelatos) mir-27 969 miR-27a(RFAM- TTCACAGTGGCTAAGTTCCGCC 1188 (Mourelatos) Human) mir-92_2 970miR-92 (RFAM-M. mu.) TATTGCACTTGTCCCGGCCTG 1182 mir-92_2 970 mir-92TATTGCACTTGTCCCGGCCTGT 216 mir-93 971 miR-93 AAAGTGCTGTTCGTGCAGGTAG 1189(Mourelatos) (Mourelatos) mir-93 971 miR-93 (Tuschl) CAAAGTGCTGTTCGTGC1190 (Mourelatos) mir-93 971 miR-93 (RFAM-M. mu.)CAAAGTGCTGTTCGTGCAGGTAG 1191 (Mourelatos) mir-95 972 miR-95TTCAACGGGTATTTATTGAGCA 1192 (Mourelatos) (Mourelatos) mir-99 973 miR-99AACCCGTAGATCCGATCTTGTG 1193 (Mourelatos) (Mourelatos) mir-99 973 miR-99a(Tuschl) ACCCGTAGATCCGATCTTGT 1194 (Mourelatos) mir-25 974 miR-25(Tuschl) CATTGCACTTGTCTCGGTCTGA 1195 mir-28 975 miR-28 (Tuschl)AAGGAGCTCACAGTCTATTGAG 1196 mir-31 976 miR-31 (RFAM-M. mu.)AGGCAAGATGCTGGCATAGCTG 1197 mir-31 976 miR-31 (Tuschl)GGCAAGATGCTGGCATAGCTG 1198 mir-32 977 miR-32 (Tuschl)TATTGCACATTACTAAGTTGC 1199 mir-149 978 miR-149 TCTGGCTCCGTGTCTTCACTCC1200 mir-30c_2 979 mir-30c TGTAAACATCCTACACTCTCAGC 280 mir-30c_2 979mir-30c_Ruvkun TGTAAACATCCTACACTCTCAGCT 1129 mir-99b 980 miR-99bCACCCGTAGAACCGACCTTGCG 1201 MiR-125a 981 miR-125aTCCCTGAGACCCTTTAACCTGTG 1202 MiR-125b_2 982 mir-125bTCCCTGAGACCCTAACTTGTGA 258 mir-26a_2 983 miR-26a (MichaelTTCAAGTAATCCAGGATAGGC 1203 et al) mir-26a_2 983 mir-26aTTCAAGTAATCCAGGATAGGCT 226 mir-127 984 mir-127_RuvkunTCGGATCCGTCTGAGCTTGG 1204 mir-127 984 miR-127 TCGGATCCGTCTGAGCTTGGCT1205 mir-136 985 miR-136 ACTCCATTTGTTTTGATGATGGA 1206 mir-154 986miR-154 TAGGTTATCCGTGTTGCCTTCG 1207 mir-26a_1 987 miR-26a (MichaelTTCAAGTAATCCAGGATAGGC 1203 et al) mir-26a_1 987 mir-26aTTCAAGTAATCCAGGATAGGCT 226 mir_186 988 miR-186 CAAAGAATTCTCCTTTTGGGCTT1208 mir_198 989 mir-198 GGTCCAGAGGGGAGATAGG 1209 mir_191 990 mir-191CAACGGAATCCCAAAAGCAGCT 1210 mir_191 990 mir-191_RuvkunCAACGGAATCCCAAAAGCAGCTGT 1211 mir_206 991 mir-206 TGGAATGTAAGGAAGTGTGTGG1212 mir-94/mir-106b 992 miR-94 AAAGTGCTGACAGTGCAGAT 1213mir-94/mir-106b 992 miR-106b (RFAM-M. mu.) TAAAGTGCTGACAGTGCAGAT 1214mir_184 993 miR-184 TGGACGGAGAACTGATAAGGGT 1215 mir_195 994 miR-195TAGCAGCACAGAAATATTGGC 1216 mir_193 995 miR-193 AACTGGCCTACAAAGTCCCAG1217 mir_185 996 miR-185 TGGAGAGAAAGGCAGTTC 1218 mir_188 997 miR-188CATCCCTTGCATGGTGGAGGGT 1219 mir_197 998 miR-197a TTCACCACCTTCTCCACCCAGC1220 mir_194_1 999 miR-194 TGTAACAGCAACTCCATGTGGA 1221 mir_208 1000miR-208 ATAAGACGAGCAAAAAGCTTGT 1222 mir_194_2 1001 miR-194TGTAACAGCAACTCCATGTGGA 1221 mir_139 1002 miR-139 TCTACAGTGCACGTGTCT 1223mir-200b 1003 miR-200a (RFAM- CTCTAATACTGCCTGGTAATGATG 1224 Human)mir-200b 1003 miR-200b (Michael TAATACTGCCTGGTAATGATGA 1225 et al)mir-200b 1003 miR-200b TAATACTGCCTGGTAATGATGAC 1226 mir-200a 1004miR-200a TAACACTGTCTGGTAACGATG 1227 mir-200a 1004 miR-200a (RFAM-M. mu.)TAACACTGTCTGGTAACGATGT 1228 mir-240* (Kosik) 1005 mir-240* (Kosik)TCAAGAGCAATAACGAAAAATGT 1229 mir-232* (Kosik) 1006 mir-232* (Kosik)CTGGCCCTCTCTGCCCTTCCGT 1230 mir-227* 1007 mir-226* (Kosik)ACTGCCCCAGGTGCTGCTGG 1231 (Kosik)/mir-226* (Kosik) mir-227* 1007mir-324-3p_Ruvkun CCACTGCCCCAGGTGCTGCTGG 1232 (Kosik)/mir-226* (Kosik)mir-227* 1007 mir-227* (Kosik) CGCATCCCCTAGGGCATTGGTGT 1233(Kosik)/mir-226* (Kosik) mir-244* (Kosik) 1008 mir-244* (Kosik)TCCAGCATCAGTGATTTTGTTGA 1234 mir-224* (Kosik) 1009 mir-224* (Kosik)GCACATTACACGGTCGACCTCT 1235 mir-248* (Kosik) 1010 mir-248* (Kosik)TCTCACACAGAAATCGCACCCGTC 1236 ribosomal protein 1011 hypotheticalAGCCAGGTGCCTTCACCTGCT 1155 L5/hypothetical miRNA-168 miRNA 168_2hypothetical 1012 hypothetical AGACAAACATGCTACTCTCAC 1164 miRNA-177_3miRNA-177 mir-138_3 1013 mir-138 AGCTGGTGTTGTGAATC 256 mir-138_3 1013mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127 mir-138_4 1014 mir-138AGCTGGTGTTGTGAATC 256 mir-181b_2 1015 mir-181b AACATTCATTGCTGTCGGTGGGTT260 mir-219_1 1016 mir-219 TGATTGTCCAAACGCAATTCT 271 mir-105_2 1017miR-105 TCAAATGCTCAGACTCCTGT 1185 (Mourelatos) hypothetical miRNA 1018hypothetical TTACAGCAATCCAGTAATGAT 1138 120_2 miRNA-120 cezanne 2/ 1019hypothetical TCCTGTCAGACTTTGTTCGGT 1237 hypothetical miRNA-180 miRNA-180mir-103_2 1020 mir-103 AGCAGCATTGTACAGGGCTATGA 225 mir-147 (Sanger) 1021miR-147 (RFAM- GTGTGTGGAAATGCTTCTGC 1238 Human) mir-224 (Sanger) 1022miR-224 (RFAM- CAAGTCACTAGTGGTTCCGTTTA 1239 Human) mir-134 (Sanger) 1023miR-134 (RFAM- TGTGACTGGTTGACCAGAGGG 1240 Human) mir-146 (Sanger) 1024miR-146 (RFAM- TGAGAACTGAATTCCATGGGTT 1241 Human) mir-150 (Sanger) 1025miR-150 (RFAM- TCTCCCAACCCTTGTACCAGTG 1242 Human) mir-30e (RFAM/mmu)1026 miR-30e (RFAM-M. mu.) TGTAAACATCCTTGACTGGA 1243 mir-30e (RFAM/mmu)1026 miR-97 (Michael TGTAAACATCCTTGACTGGAAG 1244 et al) mir-296(RFAM/mmu) 1027 miR-296 (RFAM-M. mu.) AGGGCCCCCCCTCAATCCTGT 1245 mir-299(RFAM/mmu) 1028 miR-299 (RFAM-M. mu.) TGGTTTACCGTCCCACATACAT 1246mir-301 (RFAM/mmu) 1029 miR-301 (RFAM-M. mu.) CAGTGCAATAGTATTGTCAAAGC1247 mir-301 (RFAM/mmu) 1029 mir-301_Ruvkun CAGTGCAATAGTATTGTCAAAGCAT1248 mir-302 (RFAM/mmu) 1030 miR-302 (RFAM-M. mu.)TAAGTGCTTCCATGTTTTGGTGA 1249 mir-34a (RFAM/mmu) 1031 mir-34c (RFAM)AGGCAGTGTAGTTAGCTGATTG 1250 mir-34a (RFAM/mmu) 1031 miR-34a (RFAM-M.mu.) AGGCAGTGTAGTTAGCTGATTGC 1251 mir_320 1032 miR-320AAAAGCTGGGTTGAGAGGGCGAA 1252 mir-321_1 1033 miR-321-1TAAGCCAGGGATTGTGGGTTC 1253 mir-135b (Ruvkun) 1034 mir-135b (Ruvkun)TATGGCTTTTCATTCCTATGTG 1254 mir-151* (Ruvkun) 1035 mir-151 (human)ACTAGACTGAAGCTCCTTGAGG 1255 mir-151* (Ruvkun) 1035 mir-151* (Ruvkun)TCGAGGAGCTCACAGTCTAGTA 1256 mir-340 (Ruvkun) 1036 mir-340 (Ruvkun)TCCGTCTCAGTTACTTTATAGCC 1257 mir-331 (Ruvkun) 1037 mir-331 (Ruvkun)GCCCCTGGGCCTATCCTAGAA 1258 mir_200c (RFAM) 1038 mir-200c (RFAM)AATACTGCCGGGTAATGATGGA 1259 mir_34b (RFAM) 1039 mir-34b (RFAM)AGGCAGTGTCATTAGCTGATTG 1260 mir_339_1 (RFAM) 1040 mir-339 (RFAM)TCCCTGTCCTCCAGGAGCTCA 1261 mir_339_2 (RFAM) 1041 mir-339 (RFAM)TCCCTGTCCTCCAGGAGCTCA 1261 mir-325 (Ruvkun) 1042 mir-325 (human)CCTAGTAGGTGTCCAGTAAGTGT 1262 mir-326 (Ruvkun) 1043 miR-326 (Ruvkun)CCTCTGGGCCCTTCCTCCAG 1263 mir-326 (Ruvkun) 1044 mir-326 (human)CCTCTGGGCCCTTCCTCCAGC 1264 mir-329-1 (Ruvkun) 1045 mir-329 (human)AACACACCTGGTTAACCTCTTT 1265 mir-329-2 (Ruvkun) 1046 mir-329 (human)AACACACCTGGTTAACCTCTTT 1265 mir-330 (Ruvkun) 1047 mir-330 (human)GCAAAGCACACGGCCTGCAGAGA 1266 mir-337 (Ruvkun) 1048 mir-337 (human)TCCAGCTCCTATATGATGCCTTT 1267 mir-345 (Ruvkun) 1049 mir-345 (human)TGCTGACTCCTAGTCCAGGGC 1268 mir-346 (Ruvkun) 1050 mir-346 (human)TGTCTGCCCGCATGCCTGCCTCT 1269 mir-187 1051 miR-187 (RFAM-TCGTGTCTTGTGTTGCAGCCG 1270 Human) mir-187 1051 mir-187TCGTGTCTTGTGTTGCAGCCGG 276 miR-24-1 1052 miR-189 (RFAM-GTGCCTACTGAGCTGATATCAGT 1271 Human) miR-24-1 1052 mir-24TGGCTCAGTTCAGCAGGAACAG 264 mir-215 1053 mir-215 ATGACCTATGAATTGACAGAC278

A list of mouse pri-miRNAs and the mature miRNAs predicted to derivefrom them is shown in Table 61. “Pri-miRNA name” indicates the gene namefor each of the pri-miRNAs. Also given in table 61 are the name andsequence of the mature miRNA derived from the pri-miRNA. Mature miRNAsequences from pri-miRNA precursors have been proposed by severalgroups; consequently, for a given pri-miRNA sequence, several miRNAs maybe disclosed and given unique names, and thus a given pri-miRNA sequencemay occur repeatedly in the table. The sequences are written in the 5′to 3′ direction and are represented in the DNA form. It is understoodthat a person having ordinary skill in the art would be able to convertthe sequence of the targets to their RNA form by simply replacing thethymidine (T) with uracil (U) in the sequence.

TABLE 61 Mouse pri-miRNA sequences and the corresponding mature miRNAsSEQ ID SEQ ID Pri-miRNA name NO Mature miRNA name Mature miRNA sequenceNO mir-26b 1273 miR-99 TTCAAGTAATTCAGGATAGGT 1147 (Mourelatos) mir-26b1273 miR-199-as TTCAAGTAATTCAGGATAGGTT 281 mir-30a 1274 miR-199-asCTTTCAGTCGGATGTTTGCAGC 193 mir-30a 1274 miR-26b (RFAM-TGTAAACATCCTCGACTGGAAGC 1184 Human) mir-30c_1 1275 miR-32 (Tuschl)TGTAAACATCCTACACTCTCAGC 280 mir-30c_1 1275 let-7c_RuvkunTGTAAACATCCTACACTCTCAGCT 1129 mir-128a 1276 mir-214TCACAGTGAACCGGTCTCTTT 1073 mir-128a 1276 miR-29b (RFAM-TCACAGTGAACCGGTCTCTTTT 200 Human) mir-29b_1 1277 mir-196TAGCACCATTTGAAATCAGT 1172 mir-29b_1 1277 hypotheticalTAGCACCATTTGAAATCAGTGT 1173 miRNA-079 mir-29b_1 1277 mir-30cTAGCACCATTTGAAATCAGTGTT 195 mir-29c 1278 mir-131_RuvkunCTAGCACCATTTGAAATCGGTT 232 mir-29c 1278 hypotheticalTAGCACCATTTGAAATCGGTTA 1100 miRNA-033 mir-123/mir-126 1279 mir-326(rodent) CATTATTACTTTTGGTACGCG 205 mir-123/mir-126 1279 mir-126TCGTACCGTGAGTAATAATGC 1076 mir-130a 1280 mir-23a CAGTGCAATGTTAAAAGGGC233 mir-130a 1280 hypothetical CAGTGCAATGTTAAAAGGGCAT 1101 miRNA-040mir-1d_1 1281 mir-132 TGGAATGTAAAGAAGTATGTA 1083 mir-1d_1 1281 mir-124a(Kosik) TGGAATGTAAAGAAGTATGTAT 213 mir-1d_1 1281 miR-200bTGGAATGTAAAGAAGTATGTATT 1134 mir-124a_3 1282 mir-100TAAGGCACGCGGTGAATGCCA 1104 mir-124a_3 1282 mir-212TTAAGGCACGCGGTGAATGCCA 235 mir-124a_3 1282 let-7aTTAAGGCACGCGGTGAATGCCAA 1105 mir-133a_2 1283 miR-189 (RFAM-TTGGTCCCCTTCAACCAGCTGT 255 Human) mir-124a_2 1284 mir-181cTAAGGCACGCGGTGAATGCCA 1104 mir-124a_2 1284 mir-108TTAAGGCACGCGGTGAATGCCA 235 mir-124a_2 1284 mir-239* (Kosik)TTAAGGCACGCGGTGAATGCCAA 1105 mir-131_1/mir-9 1285 mir-325 (rodent)TAAAGCTAGATAACCGAAAGT 211 mir-131_1/mir-9 1285 mir-19bTAAAGCTAGATAACCGAAAGTA 1080 mir-131_1/mir-9 1285 mir-124a_RuvkunTCTTTGGTTATCTAGCTGTATGA 1081 mir-15b 1286 mir-152 TAGCAGCACATCATGGTTTAC1115 mir-15b 1286 hypothetical TAGCAGCACATCATGGTTTACA 246 miRNA-111mir-16_3 1287 miR-104 TAGCAGCACGTAAATATTGGCG 196 (Mourelatos) mir-16_31287 mir-128a TAGCAGCACGTAAATATTGGCGT 1176 mir-137 1288 mir-30bTATTGCTTAAGAATACGCGTAG 270 mir-101_1 1289 mir-18 TACAGTACTGTGATAACTGA265 mir-101_1 1289 mir-128b TACAGTACTGTGATAACTGAAG 1170 mir-29a 1291miR-27a (RFAM-M. mu.) CTAGCACCATCTGAAATCGGTT 247 mir-29a 1291 mir-153TAGCACCATCTGAAATCGGTTA 1116 mir-29b_2 1292 mir-138_RuvkunTAGCACCATTTGAAATCAGT 1172 mir-29b_2 1292 hypotheticalTAGCACCATTTGAAATCAGTGT 1173 miRNA-075 mir-29b_2 1292 miR-30a-sTAGCACCATTTGAAATCAGTGTT 195 mir-148a 1293 miR-1d (Tuschl)TCAGTGCACTACAGAACTTTGT 288 mir-141 1294 mir-16_RuvkunAACACTGTCTGGTAAAGATGG 261 mir-131_3/mir-9 1295 mir-124a (Kosik)TAAAGCTAGATAACCGAAAGT 211 mir-131_3/mir-9 1295 mir-7b (rodent)TAAAGCTAGATAACCGAAAGTA 1080 mir-131_3/mir-9 1295 mir-19aTCTTTGGTTATCTAGCTGTATGA 1081 mir-23a 1296 miR-1 (RFAM)ATCACATTGCCAGGGATTTCC 289 mir-24_2 1297 mir-124a_RuvkunTGGCTCAGTTCAGCAGGAACAG 264 mir-140 1298 miR-199b (mouse)AGTGGTTTTACCCTATGGTAG 192 mir-140 1298 mir-205 TACCACAGGGTAGAACCACGGA1065 mir-140 1298 mir-26b TACCACAGGGTAGAACCACGGACA 1066 let-7a_4 1299mir-16_Ruvkun TGAGGTAGTAGGTTGTATAGTT 222 mir-125b_1 1300 mir-131_RuvkunTCCCTGAGACCCTAACTTGTGA 258 mir-26a_1 1301 mir-29b TTCAAGTAATCCAGGATAGGC1203 mir-26a_1 1301 hypothetical TTCAAGTAATCCAGGATAGGCT 226 miRNA-154let-7i 1302 hypothetical TGAGGTAGTAGTTTGTGCT 209 miRNA-179 let-7i 1302miR-1d (Tuschl) TGAGGTAGTAGTTTGTGCTGTT 1078 mir-21 1303 mir-125bTAGCTTATCAGACTGATGTTGA 236 mir-22 1304 mir-131 AAGCTGCCAGTTGAAGAACTGT215 mir-142 1305 mir-131_Ruvkun CATAAAGTAGAAAGCACTAC 217 mir-142 1305hypothetical TGTAGTGTTTCCTACTTTATGG 1086 miRNA-105 mir-142 1305 mir-218TGTAGTGTTTCCTACTTTATGGA 1087 mir-144 1306 mir-26a TACAGTATAGATGATGTACTAG237 mir-152 1307 miR-99a (Tuschl) TCAGTGCATGACAGAACTTGG 282 mir-153_21308 mir-29c TTGCATAGTCACAAAAGTGA 201 let-7a_1 1309 mir-16TGAGGTAGTAGGTTGTATAGTT 222 let-7d 1310 mir-144 AGAGGTAGTAGGTTGCATAGT 245let-7d 1310 hypothetical AGAGGTAGTAGGTTGCATAGTT 1113 miRNA-171 let-7d1310 miR-204 (Tuschl) CTATACGACCTGCTGCCTTTCT 1114 let-7f_1 1311 miR-9TGAGGTAGTAGATTGTATAGT 1098 let-7f_1 1311 hypotheticalTGAGGTAGTAGATTGTATAGTT 231 miRNA-138 mir-23b 1312 mir-1dATCACATTGCCAGGGATTACCAC 208 miR-24-1 1313 mir-124a (Kosik)GTGCCTACTGAGCTGATATCAGT 1271 miR-24-1 1313 hypotheticalTGGCTCAGTTCAGCAGGAACAG 264 miRNA-070 mir-27b 1314 miR-29c (Tuschl)TTCACAGTGGCTAAGTTCTG 202 mir-27b 1314 mir-135 TTCACAGTGGCTAAGTTCTGC 1059mir-131_2/mir-9 1315 mir-107 TAAAGCTAGATAACCGAAAGT 211 mir-131_2/mir-91315 miR-224 (RFAM- TAAAGCTAGATAACCGAAAGTA 1080 mouse) mir-131_2/mir-91315 mir-124a TCTTTGGTTATCTAGCTGTATGA 1081 mir-15a 1316 miR-20 (RFAM-TAGCAGCACATAATGGTTTGT 1151 Human) mir-15a 1316 miR-92 (RFAM-TAGCAGCACATAATGGTTTGTG 269 M. mu.) mir-16_1 1317 mir-98TAGCAGCACGTAAATATTGGCG 196 mir-16_1 1317 mir-30c_RuvkunTAGCAGCACGTAAATATTGGCGT 1176 mir-124a_1 1318 miR-132 (RFAM-TAAGGCACGCGGTGAATGCCA 1104 Human) mir-124a_1 1318 miR-140-asTTAAGGCACGCGGTGAATGCCA 235 mir-124a_1 1318 hypotheticalTTAAGGCACGCGGTGAATGCCAA 1105 miRNA-181 mir-18 1319 mir-124aTAAGGTGCATCTAGTGCAGATA 262 mir-18 1319 miR-27 TAAGGTGCATCTAGTGCAGATAG1177 (Mourelatos) mir-20 1320 mir-23b TAAAGTGCTTATAGTGCAGGTA 1126 mir-201320 mir-199a TAAAGTGCTTATAGTGCAGGTAG 254 mir-30b 1321 miR-31 (Tuschl)TGTAAACATCCTACACTCAGC 266 mir-30b 1321 mir-18_RuvkunTGTAAACATCCTACACTCAGCT 1137 mir-30d 1322 miR-186 TGTAAACATCCCCGACTGGAAG240 mir-30d 1322 let-7i_Ruvkun TGTAAACATCCCCGACTGGAAGCT 1108 let-7b 1323mir-135 TGAGGTAGTAGGTTGTGTGGTT 212 let-7b 1323 mir-133aTGAGGTAGTAGGTTGTGTGGTTT 1082 let7c_2 1324 let-7d* (RFAM-M. mu.)TGAGGTAGTAGGTTGTATGGTT 250 let7c_2 1324 hypotheticalTGAGGTAGTAGGTTGTATGGTTT 1120 miRNA-170 let-7c_1 1325 let-7dTGAGGTAGTAGGTTGTATGGTT 250 let-7c_1 1325 miR-135 (RFAM-TGAGGTAGTAGGTTGTATGGTTT 1120 Human) mir-99 1326 miR-203 (Tuschl)AACCCGTAGATCCGATCTTGTG 1193 (Mourelatos) mir-99 1326 mir-34ACCCGTAGATCCGATCTTGT 1194 (Mourelatos) LOC 114614 1327 mir-187TTAATGCTAATTGTGATAGGGG 1459 containing miR- 155/ hypothetical miRNA-071let-7e 1328 let-7a TGAGGTAGGAGGTTGTATAGT 249 mir-1d_2 1329 miR-10b(Michael TGGAATGTAAAGAAGTATGTA 1083 et al) mir-1d_2 1329 miR-139TGGAATGTAAAGAAGTATGTAT 213 mir-1d_2 1329 mir-124aTGGAATGTAAAGAAGTATGTATT 1134 mir-133a_1 1330 mir-24TTGGTCCCCTTCAACCAGCTGT 255 mir-143 1331 miR-15b (MichaelTGAGATGAAGCACTGTAGCTC 1088 et al) mir-143 1331 mir-253* (Kosik)TGAGATGAAGCACTGTAGCTCA 220 mir-145 1332 mir-148b GTCCAGTTTTCCCAGGAATCC1122 mir-145 1332 let-7f GTCCAGTTTTCCCAGGAATCCCTT 252 mir-122a 1333miR-172 (RFAM-M. mu.) TGGAGTGTGACAATGGTGTTTG 1084 mir-122a 1333mir-124a_Ruvkun TGGAGTGTGACAATGGTGTTTGT 214 mir-19b_2 1334 mir-22TGTGCAAATCCATGCAAAACTGA 241 let-7f_2 1335 hypotheticalTGAGGTAGTAGATTGTATAGT 1098 miRNA-137 let-7f_2 1335 mir-131TGAGGTAGTAGATTGTATAGTT 231 mir-26a_2 1336 mir-29a_RuvkunTTCAAGTAATCCAGGATAGGC 1203 mir-26a_2 1336 hypotheticalTTCAAGTAATCCAGGATAGGCT 226 miRNA-153 mir-127 1337 mir-103TCGGATCCGTCTGAGCTTGG 1204 mir-127 1337 mir-17as/mir-91TCGGATCCGTCTGAGCTTGGCT 1205 mir-136 1338 mir-91_RuvkunACTCCATTTGTTTTGATGATGGA 1206 mir-154 1339 mir-17-3p (mouse)TAGGTTATCCGTGTTGCCTTCG 1207 mir-149 1340 let-7gL_RuvkunTCTGGCTCCGTGTCTTCACTCC 1200 mir-30c_2 1341 miR-31 (RFAM-TGTAAACATCCTACACTCTCAGC 280 M. mu.) mir-30c_2 1341 let-7cTGTAAACATCCTACACTCTCAGCT 1129 mir-99b 1342 mir-101b (rodent)CACCCGTAGAACCGACCTTGCG 1201 MiR-125a 1343 mir-106 (mouse)TCCCTGAGACCCTTTAACCTGTG 1202 MiR-125b_2 1344 miR-9TCCCTGAGACCCTAACTTGTGA 258 mir-221 1345 miR-200a (RFAM-AGCTACATTGTCTGCTGGGTTT 1106 Human) mir-221 1345 miR-26a (MichaelAGCTACATTGTCTGCTGGGTTTC 238 et al) mir-203 1346 mir-10bGTGAAATGTTTAGGACCACTAG 197 mir-203 1346 mir-128 (Kosik)TGAAATGTTTAGGACCACTAG 1068 mir-203 1346 mir-204 TGAAATGTTTAGGACCACTAGA1069 let-7g 1347 hypothetical TGAGGTAGTAGTTTGTACAGT 285 miRNA-176 let-7g1347 mir-1d TGAGGTAGTAGTTTGTACAGTT 1152 mir-101_3 1348 miR-200aTACAGTACTGTGATAGCTGAAG 1460 mir-106 1349 miR-200a (RFAM-CAAAGTGCTAACAGTGCAGGTA 1461 M. mu.) mir-17/mir-91 1350 mir-123/mir-126asACTGCAGTGAGGGCACTTGT 1462 mir-17/mir-91 1350 mir-227* (Kosik)CAAAGTGCTTACAGTGCAGGTAG 1181 mir-17/mir-91 1350 miR-195CAAAGTGCTTACAGTGCAGGTAGT 204 mir-199b 1351 mir-226* (Kosik)CCCAGTGTTTAGACTACCTGTTC 1463 mir-199b 1351 mir-217 (rodent)TACAGTAGTCTGCACATTGGTT 1118 hypothetical 1352 mir-324-3p_RuvkunAGAGGTATAGCGCATGGGAAGA 1464 miRNA 105 hypothetical 1352 miR-127TTCCTATGCATATACTTCTTT 1132 miRNA 105 mir-211 1353 mir-244* (Kosik)TTCCCTTTGTCATCCTTTGCCT 1465 mir-217 1354 mir-224* (Kosik)TACTGCATCAGGAACTGACTGGAT 1466 mir-224 (Sanger) 1355 mir-248* (Kosik)TAAGTCACTAGTGGTTCCGTTTA 1467 mir-7_3 1356 mir-138 TGGAAGACTTGTGATTTTGTT1468 mir-325 (Ruvkun) 1357 mir-138_Ruvkun CCTAGTAGGTGCTCAGTAAGTGT 1469mir-326 (Ruvkun) 1358 mir-181b CCTCTGGGCCCTTCCTCCAG 1263 mir-326(Ruvkun) 1358 miR-298 CCTCTGGGCCCTTCCTCCAGT 1470 mir-329-1 1359 mir-103AACACACCCAGCTAACCTTTTT 1471 (Ruvkun) mir-330 (Ruvkun) 1360 miR-134(RFAM- GCAAAGCACAGGGCCTGCAGAGA 1472 Human) mir-337 (Ruvkun) 1361 miR-146(RFAM- TTCAGCTCCTATATGATGCCTTT 1473 Human) mir-345 (Ruvkun) 1362 miR-30e(RFAM- TGCTGACCCCTAGTCCAGTGC 1474 M. mu.) mir-346 (Ruvkun) 1363 miR-97(Michael TGTCTGCCCGAGTGCCTGCCTCT 1475 et al) mir-151* (Ruvkun) 1364miR-193 ACTAGACTGAGGCTCCTTGAGG 1476 mir-151* (Ruvkun) 1364 mir-340(Ruvkun) CTAGACTGAGGCTCCTTGAGG 1477 mir-151* (Ruvkun) 1364 miR-299(RFAM- TCGAGGAGCTCACAGTCTAGTA 1256 M. mu.) mir_34b (RFAM) 1365 mir-331(Ruvkun) TAGGCAGTGTAATTAGCTGATTG 1478 glutamate 1366 miR-143 (MichaelTGTTATAGTATTCCACCTACC 1060 receptor, et al) ionotrophic, AMPA3/hypothetical miRNA-033 mir-34 1367 mir-138 TGGCAGTGTCTTAGCTGGTTGT 194mir-34 1367 mir-30a TGGCAGTGTCTTAGCTGGTTGTT 1067 mir-7_1/mir-7_1* 1368mir-191_Ruvkun CAACAAATCACAGTCTGCCATA 1070 mir-7_1/mir-7_1* 1368 mir-29bTGGAAGACTAGTGATTTTGTT 198 mir-10b 1369 mir-210 CCCTGTAGAACCGAATTTGTGT1071 mir-10b 1369 miR-29b (RFAM- TACCCTGTAGAACCGAATTTGT 199 M. mu.)mir-10b 1369 mir-34b (mouse) TACCCTGTAGAACCGAATTTGTG 1072 mir-132 1370mir-130a TAACAGTCTACAGCCATGGTCG 1077 mir-132 1370 miR-196 (Tuschl)TAACAGTCTACAGCCATGGTCGC 206 mir-108_1 1371 mir-130 (Kosik)ATAAGGATTTTTAGGGGCATT 207 mir-212 1372 miR-1 (RFAM)TAACAGTCTCCAGTCACGGCC 210 hypothetical 26 mir-143 TGGGCAAGAGGACTTTTTAAT1079 miRNA 023 mir-214 37 mir-15b ACAGCAGGCACAGACAGGCAG 219 hypothetical43 mir-145 TGTCAACAAAACTGCTTACAA 1092 miRNA 040 hypothetical 1373miR-145 (Michael TGACAGGAAATCTTTGAGAGG 1094 miRNA 043 et al) mir-2051374 mir-101 TCCTTCATTCCACCGGAGTCTG 224 mir-33a 1375 miR-29b (RFAM-GTGCATTGTAGTTGCATTG 227 M. mu.) mir-196_2 1376 mir-7-1*_RuvkunTAGGTAGTTTCATGTTGTTGG 1097 mir-196_2 1376 mir-148aTAGGTAGTTTCATGTTGTTGGG 228 hypothetical 1377 mir-122aTTGCATGCCCTATTGATTCTC 1099 miRNA 055 hypothetical 1378 miR-122a, bTGTCAGATGCTTAATGTTCTT 1102 miRNA 058 (Tuschl) mir-218_1 1379 mir-140TTGTGCTTGATCTAACCATGT 234 mir-218_1 1379 mir-196 TTGTGCTTGATCTAACCATGTG1103 mir-222 1380 miR-200b (Michael AGCTACATCTGGCTACTGGGTCT 1107 et al)mir-222 1380 let-7i AGCTACATCTGGCTACTGGGTCTC 239 mir-128b 1381 mir-142TCACAGTGAACCGGTCTCTTT 1073 mir-128b 1381 hypotheticalTCACAGTGAACCGGTCTCTTTC 242 miRNA-023 mir-219_2 1382 mir-30b_RuvkunTGATTGTCCAAACGCAATTCT 271 hypothetical 1383 mir-19bTCACATTTGCCTGCAGAGATT 1109 miRNA 070 mir-129_2 1384 miR-196 (Tuschl)AAGCCCTTACCCCAAAAAGCAT 1110 mir-129_2 1384 mir-128 (Kosik)CTTTTTGCGGTCTGGGCTTGC 243 mir-129_2 1384 miR-142-asCTTTTTGCGGTCTGGGCTTGCT 1111 mir-133b 1385 miR-142asTTGGTCCCCTTCAACCAGCTA 244 (Michael et al) hypothetical 78 let-7fTGGTTAAAATATTAATGGGGC 1112 miRNA 075 hypothetical 1386 let-7f (MichaelTGATATGTTTGATATTGGG 1117 miRNA 079 et al) mir-204 1387 let-7d_RuvkunTTCCCTTTGTCATCCTATGCCT 251 mir-204 1387 miR-10b (Tuschl)TTCCCTTTGTCATCCTATGCCTG 1121 mir-213/mir- 1388 mir-137AACATTCAACGCTGTCGGTGAG 1096 181a_2 mir-213/mir- 1388 hypotheticalAACATTCAACGCTGTCGGTGAGT 223 181a_2 miRNA-043 mir-213/mir- 1388 let-7f(Michael ACCATCGACCGTTGATTGTACC 253 181a_2 et al) hypothetical 1389mir-26a TAGGCCAAATGGCGCATCAAT 1124 miRNA 090 mir-138_2 1390 mir-92AGCTGGTGTTGTGAATC 256 mir-138_2 1390 miR-27* (MichaelAGCTGGTGTTGTGAATCAGGCCG 1127 et al) mir-196_1 1391 miR-29b (RFAM-TAGGTAGTTTCATGTTGTTGG 1097 Human) mir-196_1 1391 mir-7TAGGTAGTTTCATGTTGTTGGG 228 mir-199a_2 1392 miR-202 (mouse)CCCAGTGTTCAGACTACCTGTT 1128 mir-199a_2 1392 mir-15aCCCAGTGTTCAGACTACCTGTTC 259 mir-199a_2 1392 mir-211 (rodent)TACAGTAGTCTGCACATTGGTT 1118 mir-181b_1 1393 mir-16AACATTCATTGCTGTCGGTGGGTT 260 hypothetical 1394 miR-26a (MichaelTGACAGTCAATTAACAAGTTT 1130 miRNA 101 et al) hypothetical 1395mir-127_Ruvkun TTCCTCCTCCTCCGACTCGGA 1135 miRNA 111 mir-218_2 1396mir-33a TTGTGCTTGATCTAACCATGT 234 mir-218_2 1396 mir-24TTGTGCTTGATCTAACCATGTG 1103 mir-148b 1397 mir-30d TCAGTGCATCACAGAACTTTGT272 mir-216 1398 mir-30d_Ruvkun TAATCTCAGCTGGCAACTGTG 274 hypothetical1399 miR-136 TAAACTGGCTGATAATTTTTG 1141 miRNA 137 hypothetical 1400miR-154 TGCAAGTATGAAAATGAGATT 1142 miRNA 138 mir-210 1401 let-7cCTGTGCGTGTGACAGCGGCTG 277 mir-223 1402 let-7c_RuvkunTGTCAGTTTGTCAAATACCCC 279 hypothetical 1403 miR-149TGCAAGCAGATGCTGATAATA 1145 miRNA 153 hypothetical 1404 mir-30cTTAAAGTGGATGTGTGTTATT 1146 miRNA 154 mir-135_1 1405 hypotheticalTATGGCTTTTTATTCCTATGTGA 1149 miRNA-101 mir-135_1 1405 let-7eTATGGCTTTTTATTCCTATGTGAT 283 non-coding RNA in 1406 mir-181bTATGGCTTTTTATTCCTATGTGA 1149 rhabdomyosarcoma/ mir-135_2 non-coding RNAin 1406 miR-155/ TATGGCTTTTTATTCCTATGTGAT 283 rhabdomyosarcoma/hypothetical mir-135_2 miRNA-071 hypothetical 1407 mir-30c_RuvkunTGATCTTGCTCTAACACTTGG 1157 miRNA 170 glutamate 174 miR-99bTGACAAGTATGTTTTATCGTT 1158 receptor, ionotropic, AMPA 2/hypotheticalmiRNA-171 hypothetical 179 miR-125a TAGGAGTTTGATATGACATAT 1163 miRNA 176hypothetical 1408 mir-125b TGAAAGGCACTTTGTCCAATT 1166 miRNA 179hypothetical 1409 mir-221 TCACCTGCTCTGGAAGTAGTT 1167 miRNA 181 mir-181c1410 mir-133a AACATTCAACCTGTCGGTGAGT 290 mir-100_1 1411 let-7bAACCCGTAGATCCGAACTTGTG 275 mir-103_1 950 mir-29a AGCAGCATTGTACAGGGCTATGA225 mir-107 1412 mir-141 AGCAGCATTGTACAGGGCTATCA 229 mir-19a 1413 mir-20TGTGCAAATCTATGCAAAACTGA 268 mir-19b_1 1414 mir-21AGTTTTGCAGGTTTGCATCCAGC 1179 mir-19b_1 1414 mir-223TGTGCAAATCCATGCAAAACTGA 241 mir-92_1 1415 hypotheticalTATTGCACTTGTCCCGGCCTG 1182 miRNA-090 mir-92_1 1415 miR-9TATTGCACTTGTCCCGGCCTGT 216 mir-98 1416 mir-131 TGAGGTAGTAAGTTGTATTGTT257 mir-104 1417 mir-221 (RFAM- TCAACATCAGTCTGATAAGCTA 335 (Mourelatos)mmu) mir-27 1418 mir-213 TTCACAGTGGCTAAGTTCC 1186 (Mourelatos) mir-271418 mir-222 (RFAM- TTCACAGTGGCTAAGTTCCGC 1187 (Mourelatos) mmu) mir-271418 mir-203 TTCACAGTGGCTAAGTTCCGCC 1188 (Mourelatos) mir-31 1419mir-178 (Kosik) AGGCAAGATGCTGGCATAGCTG 1197 mir-31 1419 miR-203 (RFAM-GGCAAGATGCTGGCATAGCTG 1198 M. mu.) mir-32 1420 let-7gTATTGCACATTACTAAGTTGC 1199 mir_186 1421 miR-326 (Ruvkun)CAAAGAATTCTCCTTTTGGGCTT 1208 mir_191 1422 mir-329 (mouse)CAACGGAATCCCAAAAGCAGCT 1210 mir_191 1422 miR-27a (RFAM-CAACGGAATCCCAAAAGCAGCTGT 1211 Human) mir_195 1423 mir-330 (rodent)TAGCAGCACAGAAATATTGGC 1216 mir_193 1424 mir-337 (rodent)AACTGGCCTACAAAGTCCCAG 1217 mir_188 1425 mir-345 (rodent)CATCCCTTGCATGGTGGAGGGT 1219 mir_208 1426 mir-346 (mouse)ATAAGACGAGCAAAAAGCTTGT 1222 mir_139 1427 mir-151* (Ruvkun)TCTACAGTGCACGTGTCT 1223 mir-200b 1428 mir-151 (rodent)CTCTAATACTGCCTGGTAATGATG 1224 mir-200b 1428 mir-216TAATACTGCCTGGTAATGATGA 1225 mir-200b 1428 mir-219TAATACTGCCTGGTAATGATGAC 1226 mir-200a 1429 mir-181aTAACACTGTCTGGTAACGATG 1227 mir-200a 1429 mir-151L (rodent)TAACACTGTCTGGTAACGATGT 1228 mir-227* 1430 mir-191 ACTGCCCCAGGTGCTGCTGG1231 (Kosik)/mir-226* (Kosik) mir-227* 1430 hypotheticalCCACTGCCCCAGGTGCTGCTGG 1232 (Kosik)/mir-226* miRNA-058 (Kosik) mir-227*1430 hypothetical CGCATCCCCTAGGGCATTGGTGT 1233 (Kosik)/mir-226*miRNA-055 (Kosik) mir-244* (Kosik) 1431 mir-218 TCCAGCATCAGTGATTTTGTTGA1234 mir-224* (Kosik) 1432 mir-253* (Kosik) GCACATTACACGGTCGACCTCT 1235mir-248* (Kosik) 1433 mir-222 TCTCACACAGAAATCGCACCCGTC 1236 mir-138_31434 mir-19b* (Michael AGCTGGTGTTGTGAATC 256 et al) mir-138_3 1434mir-27b AGCTGGTGTTGTGAATCAGGCCG 1127 mir-181b_2 1435 mir-15_RuvkunAACATTCATTGCTGTCGGTGGGTT 260 mir-103_2 1436 miR-101 (RFAM-AGCAGCATTGTACAGGGCTATGA 225 Human) mir-134 (Sanger) 1437 mir-129TGTGACTGGTTGACCAGAGGG 1240 mir-146 (Sanger) 1438 mir-129as/mir-TGAGAACTGAATTCCATGGGTT 1241 258* (Kosik) mir-30e 1439 miR-129b (RFAM-TGTAAACATCCTTGACTGGA 1243 (RFAM/mmu) Human) mir-30e 1439 miR-135 (RFAM-TGTAAACATCCTTGACTGGAAG 1244 (RFAM/mmu) Human) mir-299 1440 mir-133bTGGTTTACCGTCCCACATACAT 1246 (RFAM/mmu) mir-340 (Ruvkun) 1441 miR-188TCCGTCTCAGTTACTTTATAGCC 1257 mir-331 (Ruvkun) 1442 miR-208GCCCCTGGGCCTATCCTAGAA 1258 mir-187 1443 miR-199-s TCGTGTCTTGTGTTGCAGCCG1270 mir-187 1443 let-7b_Ruvkun TCGTGTCTTGTGTTGCAGCCGG 276 miR-201 1444miR-187 (RFAM- TACTCAGTAAGGCATTGTTCT 1479 Human) miR-207 1445 miR-201GCTTCTCCTGGCTCTCCTCCCTC 1480 miR-291 1446 miR-291AAAGTGCTTCCACTTTGTGTGCC 1481 miR-291 1446 miR-207 CATCAAAGTGGAGGCCCTCTCT1482 miR-292 1447 miR-291 AAGTGCCGCCAGGTTTTGAGTGT 1483 miR-292 1447miR-292 ACTCAAACTGGGGGCTCTTTTG 1484 miR-293 1448 miR-292AGTGCCGCAGAGTTTGTAGTGT 1485 miR-294 1449 miR-293 AAAGTGCTTCCCTTTTGTGTGT1486 miR-295 1450 miR-294 AAAGTGCTACTACTTTTGAGTCT 1487 miR-300 1451miR-295 TATGCAAGGGCAAGCTCTCTTC 1488 miR-322 1452 miR-300AAACATGAAGCGCTGCAACA 1489 miR-344 1453 miR-322 TGATCTAGCCAAAGCCTGACTGT1490 miR-350 1454 miR-344 TTCACAAAGCCCATACACTTTCAC 1491 miR-290 1455miR-350 CTCAAACTATGGGGGCACTTTTT 1492 miR-351 1456 miR-290TCCCTGAGGAGCCCTTTGAGCCTG 1493 miR-341 1457 miR-351 TCGATCGGTCGGTCGGTCAGT1494 miR-298 1458 miR-341 GGCAGAGGAGGGCTGTTCTTCC 1495

A list of rat pri-miRNAs and the mature miRNAs predicted to derive fromthem is shown in Table 62. “Pri-miRNA name” indicates the gene name foreach of the pri-miRNAs. Also given in table 62 are the name and sequenceof the mature miRNA derived from the pri-miRNA. Mature miRNA sequencesfrom pri-miRNA precursors have been proposed by several groups;consequently, for a given pri-miRNA sequence, several miRNAs may bedisclosed and given unique names, and thus a given pri-miRNA sequencemay occur repeatedly in the table. The sequences are written in the 5′to 3′ direction and are represented in the DNA form. It is understoodthat a person having ordinary skill in the art would be able to convertthe sequence of the targets to their RNA form by simply replacing thethymidine (T) with uracil (U) in the sequence.

TABLE 62 Rat pri-miRNA sequences and the corresponding mature miRNAs SEQSEQ ID ID Pri-miRNA name NO Mature miRNA name Mature miRNA sequence NOmir-20 1496 miR-20* (mouse) ACTGCATTACGAGCACTTACA 1608 mir-20 1496miR-20 (RFAM- TAAAGTGCTTATAGTGCAGGTA 1126 Human) mir-20 1496 mir-20TAAAGTGCTTATAGTGCAGGTAG 254 mir-151* (Ruvkun) 1497 mir-151L (rodent)ACTAGACTGAGGCTCCTTGAGG 1476 mir-151* (Ruvkun) 1497 mir-151 (rodent)CTAGACTGAGGCTCCTTGAGG 1477 mir-151* (Ruvkun) 1497 mir-151* (Ruvkun)TCGAGGAGCTCACAGTCTAGTA 1256 mir-346 (Ruvkun) 1498 miR-346 (rat)TGTCTGCCTGAGTGCCTGCCTCT 1609 mir-143 1499 miR-143 (MichaelTGAGATGAAGCACTGTAGCTC 1088 et al) mir-143 1499 mir-143TGAGATGAAGCACTGTAGCTCA 220 mir-203 1500 mir-203 GTGAAATGTTTAGGACCACTAG197 mir-203 1500 miR-203 (RFAM-M. mu.) TGAAATGTTTAGGACCACTAG 1068mir-203 1500 miR-203 (Tuschl) TGAAATGTTTAGGACCACTAGA 1069 mir-26b 1501miR-26b (RFAM- TTCAAGTAATTCAGGATAGGT 1147 Human) mir-26b 1501 mir-26bTTCAAGTAATTCAGGATAGGTT 281 mir-128a 1276 mir-128 (Kosik)TCACAGTGAACCGGTCTCTTT 1073 mir-128a 1276 mir-128a TCACAGTGAACCGGTCTCTTTT200 mir-29b_1 1277 miR-29b (RFAM- TAGCACCATTTGAAATCAGT 1172 Human)mir-29b_1 1277 miR-29b (RFAM-M. mu.) TAGCACCATTTGAAATCAGTGT 1173mir-29b_1 1502 mir-29b TAGCACCATTTGAAATCAGTGTT 195 mir-29c 1278 mir-29cCTAGCACCATTTGAAATCGGTT 232 mir-29c 1278 miR-29c (Tuschl)TAGCACCATTTGAAATCGGTTA 1100 mir-123/mir-126 1503 mir-123/mir-126asCATTATTACTTTTGGTACGCG 205 mir-123/mir-126 1503 mir-126TCGTACCGTGAGTAATAATGC 1076 mir-130a 1504 mir-130a CAGTGCAATGTTAAAAGGGC233 mir-130a 1504 mir-130 (Kosik) CAGTGCAATGTTAAAAGGGCAT 1101 mir-124a_31282 mir-124a (Kosik) TAAGGCACGCGGTGAATGCCA 1104 mir-124a_3 1282mir-124a TTAAGGCACGCGGTGAATGCCA 235 mir-124a_3 1282 mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA 1105 mir-15b 1286 miR-15b (MichaelTAGCAGCACATCATGGTTTAC 1115 et al) mir-15b 1286 mir-15bTAGCAGCACATCATGGTTTACA 246 mir-16_3 1505 mir-16 TAGCAGCACGTAAATATTGGCG196 mir-16_3 1505 mir-16_Ruvkun TAGCAGCACGTAAATATTGGCGT 1176 mir-1371288 mir-137 TATTGCTTAAGAATACGCGTAG 270 mir-101_1 1289 mir-101TACAGTACTGTGATAACTGA 265 mir-101_1 1289 miR-101 (RFAM-TACAGTACTGTGATAACTGAAG 1170 Human) mir-29a 1291 mir-29aCTAGCACCATCTGAAATCGGTT 247 mir-29a 1291 mir-29a_RuvkunTAGCACCATCTGAAATCGGTTA 1116 mir-29b_2 1292 miR-29b (RFAM-TAGCACCATTTGAAATCAGT 1172 Human) mir-29b_2 1292 miR-29b (RFAM-M. mu.)TAGCACCATTTGAAATCAGTGT 1173 mir-29b_2 1292 mir-29bTAGCACCATTTGAAATCAGTGTT 195 mir-131_3/mir-9 1506 mir-131TAAAGCTAGATAACCGAAAGT 211 mir-131_3/mir-9 1506 mir-131_RuvkunTAAAGCTAGATAACCGAAAGTA 1080 mir-131_3/mir-9 1506 miR-9TCTTTGGTTATCTAGCTGTATGA 1081 mir-23a 1507 mir-23a ATCACATTGCCAGGGATTTCC289 mir-140 1508 mir-140 AGTGGTTTTACCCTATGGTAG 192 mir-140 1508miR-140-as TACCACAGGGTAGAACCACGGA 1065 mir-140 1508 mir-239* (Kosik)TACCACAGGGTAGAACCACGGACA 1066 mir-125b_1 1509 mir-125bTCCCTGAGACCCTAACTTGTGA 258 mir-26a_1 1510 miR-26a (MichaelTTCAAGTAATCCAGGATAGGC 1203 et al) mir-26a_1 1510 mir-26aTTCAAGTAATCCAGGATAGGCT 226 let-7i 1302 let-7i TGAGGTAGTAGTTTGTGCT 209let-7i 1302 let-7i_Ruvkun TGAGGTAGTAGTTTGTGCTGTT 1078 mir-21 1511 mir-21TAGCTTATCAGACTGATGTTGA 236 mir-22 1512 mir-22 AAGCTGCCAGTTGAAGAACTGT 215mir-142 1513 mir-142 CATAAAGTAGAAAGCACTAC 217 mir-142 1513 miR-142-asTGTAGTGTTTCCTACTTTATGG 1086 mir-142 1513 miR-142asTGTAGTGTTTCCTACTTTATGGA 1087 (Michael et al) mir-144 1514 mir-144TACAGTATAGATGATGTACTAG 237 mir-152 1515 mir-152 TCAGTGCATGACAGAACTTGG282 mir-153_2 1516 mir-153 TTGCATAGTCACAAAAGTGA 201 let-7a_1 1517 let-7aTGAGGTAGTAGGTTGTATAGTT 222 let-7d 1518 let-7d AGAGGTAGTAGGTTGCATAGT 245let-7d 1518 let-7d_Ruvkun AGAGGTAGTAGGTTGCATAGTT 1113 let-7d 1518let-7d* (RFAM-M. mu.) CTATACGACCTGCTGCCTTTCT 1114 let-7f_1 1519 let-7f(Michael TGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_1 1519 let-7fTGAGGTAGTAGATTGTATAGTT 231 miR-24-1 1313 miR-189 (RFAM-GTGCCTACTGAGCTGATATCAGT 1271 Human) miR-24-1 1313 mir-24TGGCTCAGTTCAGCAGGAACAG 264 mir-124a_1 1318 mir-124a (Kosik)TAAGGCACGCGGTGAATGCCA 1104 mir-124a_1 1318 mir-124aTTAAGGCACGCGGTGAATGCCA 235 mir-124a_1 1318 mir-124a_RuvkunTTAAGGCACGCGGTGAATGCCAA 1105 mir-18 1319 mir-18 TAAGGTGCATCTAGTGCAGATA262 mir-18 1319 mir-18_Ruvkun TAAGGTGCATCTAGTGCAGATAG 1177 mir-30b 1520mir-30b TGTAAACATCCTACACTCAGC 266 mir-30b 1520 mir-30b_RuvkunTGTAAACATCCTACACTCAGCT 1137 mir-30d 1521 mir-30d TGTAAACATCCCCGACTGGAAG240 mir-30d 1521 mir-30d_Ruvkun TGTAAACATCCCCGACTGGAAGCT 1108 let-7b1522 let-7b TGAGGTAGTAGGTTGTGTGGTT 212 let-7b 1522 let-7b_RuvkunTGAGGTAGTAGGTTGTGTGGTTT 1082 let-7e 1328 let-7e TGAGGTAGGAGGTTGTATAGT249 mir-133a_1 1330 mir-133a TTGGTCCCCTTCAACCAGCTGT 255 mir-145 1332miR-145 (Michael GTCCAGTTTTCCCAGGAATCC 1122 et al) mir-145 1332 mir-145GTCCAGTTTTCCCAGGAATCCCTT 252 mir-122a 1523 miR-122a, bTGGAGTGTGACAATGGTGTTTG 1084 (Tuschl) mir-122a 1523 mir-122aTGGAGTGTGACAATGGTGTTTGT 214 let-7f_2 1335 let-7f (MichaelTGAGGTAGTAGATTGTATAGT 1098 et al) let-7f_2 1335 let-7fTGAGGTAGTAGATTGTATAGTT 231 mir-127 1337 mir-127_RuvkunTCGGATCCGTCTGAGCTTGG 1204 mir-127 1337 miR-127 TCGGATCCGTCTGAGCTTGGCT1205 mir-136 1338 miR-136 ACTCCATTTGTTTTGATGATGGA 1206 mir-154 1339miR-154 TAGGTTATCCGTGTTGCCTTCG 1207 mir-30c_2 1341 mir-30cTGTAAACATCCTACACTCTCAGC 280 mir-30c_2 1341 mir-30c_RuvkunTGTAAACATCCTACACTCTCAGCT 1129 mir-99b 1342 miR-99bCACCCGTAGAACCGACCTTGCG 1201 MiR-125a 1524 miR-125aTCCCTGAGACCCTTTAACCTGTG 1202 mir-221 1525 mir-221 (RFAM-AGCTACATTGTCTGCTGGGTTT 1106 mmu) mir-221 1525 mir-221AGCTACATTGTCTGCTGGGTTTC 238 mir-101_3 1526 mir-101b (rodent)TACAGTACTGTGATAGCTGAAG 1460 mir-17/mir-91 1527 mir-17 (human,ACTGCAGTGAAGGCACTTGT 1180 rat) mir-17/mir-91 1527 mir-91_RuvkunCAAAGTGCTTACAGTGCAGGTAG 1181 mir-17/mir-91 1527 mir-17as/mir-91CAAAGTGCTTACAGTGCAGGTAGT 204 hypothetical 1528 hypotheticalTTCCTATGCATATACTTCTTT 1132 miRNA 105 miRNA-105 mir-211 1529 mir-211(rodent) TTCCCTTTGTCATCCTTTGCCT 1465 mir-217 1530 mir-217 (rodent)TACTGCATCAGGAACTGACTGGAT 1466 mir-7_3 1531 mir-7b (rodent)TGGAAGACTTGTGATTTTGTT 1468 mir-325 (Ruvkun) 1357 mir-325 (rodent)CCTAGTAGGTGCTCAGTAAGTGT 1469 mir-326 (Ruvkun) 1532 miR-326 (Ruvkun)CCTCTGGGCCCTTCCTCCAG 1263 mir-326 (Ruvkun) 1532 mir-326 (rodent)CCTCTGGGCCCTTCCTCCAGT 1470 mir-330 (Ruvkun) 1533 mir-330 (rodent)GCAAAGCACAGGGCCTGCAGAGA 1472 mir-337 (Ruvkun) 1361 mir-337 (rodent)TTCAGCTCCTATATGATGCCTTT 1473 mir-345 (Ruvkun) 1362 mir-345 (rodent)TGCTGACCCCTAGTCCAGTGC 1474 mir_34b (RFAM) 1365 mir-34b (mouse)TAGGCAGTGTAATTAGCTGATTG 1478 mir-34 1534 mir-34 TGGCAGTGTCTTAGCTGGTTGT194 mir-34 1534 miR-172 (RFAM-M. mu.) TGGCAGTGTCTTAGCTGGTTGTT 1067mir-7_1/mir-7_1* 1535 mir-7_1*_Ruvkun CAACAAATCACAGTCTGCCATA 1070mir-7_1/mir-7_1* 1535 mir-7 TGGAAGACTAGTGATTTTGTT 198 mir-10b 1536miR-10b (Tuschl) CCCTGTAGAACCGAATTTGTGT 1071 mir-10b 1536 mir-10bTACCCTGTAGAACCGAATTTGT 199 mir-10b 1536 miR-10b (MichaelTACCCTGTAGAACCGAATTTGTG 1072 et al) mir-132 1370 miR-132 (RFAM-TAACAGTCTACAGCCATGGTCG 1077 Human) mir-132 1370 mir-132TAACAGTCTACAGCCATGGTCGC 206 mir-212 1537 mir-212 TAACAGTCTCCAGTCACGGCC210 mir-108_1 1538 mir-108 ATAAGGATTTTTAGGGGCATT 207 hypothetical 26hypothetical TGGGCAAGAGGACTTTTTAAT 1079 miRNA 023 miRNA-023 mir-214 1539mir-214 ACAGCAGGCACAGACAGGCAG 219 hypothetical 43 hypotheticalTGTCAACAAAACTGCTTACAA 1092 miRNA 040 miRNA-040 hypothetical 1540hypothetical TGACAGGAAATCTTTGAGAGG 1094 miRNA 043 miRNA-043 mir-205 1541mir-205 TCCTTCATTCCACCGGAGTCTG 224 mir-33a 1542 mir-33aGTGCATTGTAGTTGCATTG 227 mir-196_2 1543 miR-196 (Tuschl)TAGGTAGTTTCATGTTGTTGG 1097 mir-196_2 1543 mir-196 TAGGTAGTTTCATGTTGTTGGG228 mir-218_1 1544 mir-218 TTGTGCTTGATCTAACCATGT 234 mir-218_1 1544mir-253* (Kosik) TTGTGCTTGATCTAACCATGTG 1103 mir-222 1545 mir-222 (RFAM-AGCTACATCTGGCTACTGGGTCT 1107 mmu) mir-222 1545 mir-222AGCTACATCTGGCTACTGGGTCTC 239 mir-128b 1381 mir-128 (Kosik)TCACAGTGAACCGGTCTCTTT 1073 mir-128b 1381 mir-128b TCACAGTGAACCGGTCTCTTTC242 mir-219_2 1546 mir-219 TGATTGTCCAAACGCAATTCT 271 hypothetical 1547hypothetical TCACATTTGCCTGCAGAGATT 1109 miRNA 070 miRNA-070 mir-129_21548 mir-129as/mir- AAGCCCTTACCCCAAAAAGCAT 1110 258* (Kosik) mir-129_21548 mir-129 CTTTTTGCGGTCTGGGCTTGC 243 mir-129_2 1548 miR-129b (RFAM-CTTTTTGCGGTCTGGGCTTGCT 1111 Human) mir-133b 1385 mir-133bTTGGTCCCCTTCAACCAGCTA 244 hypothetical 78 hypotheticalTGGTTAAAATATTAATGGGGC 1112 miRNA 075 miRNA-075 mir-204 1549 mir-204TTCCCTTTGTCATCCTATGCCT 251 mir-204 1549 miR-204 (Tuschl)TTCCCTTTGTCATCCTATGCCTG 1121 mir-213/mir- 1550 mir-178 (Kosik)AACATTCAACGCTGTCGGTGAG 1096 181a_2 mir-213/mir- 1550 mir-181aAACATTCAACGCTGTCGGTGAGT 223 181a_2 mir-213/mir- 1550 mir-213ACCATCGACCGTTGATTGTACC 253 181a_2 hypothetical 1551 hypotheticalTAGGCCAAATGGCGCATCAAT 1124 miRNA 090 miRNA-090 mir-138_2 1552 mir-138AGCTGGTGTTGTGAATC 256 mir-138_2 1552 mir-138_RuvkunAGCTGGTGTTGTGAATCAGGCCG 1127 mir-199a_2 1553 miR-199-sCCCAGTGTTCAGACTACCTGTT 1128 mir-199a_2 1553 mir-199aCCCAGTGTTCAGACTACCTGTTC 259 mir-199a_2 1553 miR-199-asTACAGTAGTCTGCACATTGGTT 1118 hypothetical 1554 hypotheticalTGACAGTCAATTAACAAGTTT 1130 miRNA 101 miRNA-101 mir-148b 1397 mir-148bTCAGTGCATCACAGAACTTTGT 272 mir-216 1555 mir-216 TAATCTCAGCTGGCAACTGTG274 hypothetical 1399 hypothetical TAAACTGGCTGATAATTTTTG 1141 miRNA 137miRNA-137 hypothetical 1556 hypothetical TGCAAGTATGAAAATGAGATT 1142miRNA 138 miRNA-138 mir-210 1557 mir-210 CTGTGCGTGTGACAGCGGCTG 277mir-223 1558 mir-223 TGTCAGTTTGTCAAATACCCC 279 hypothetical 1404hypothetical TTAAAGTGGATGTGTGTTATT 1146 miRNA 154 miRNA-154 non-codingRNA in 13 miR-135 (RFAM- TATGGCTTTTTATTCCTATGTGA 1149 rhabdomyosarcoma/Human) mir-135_2 non-coding RNA in 13 mir-135 TATGGCTTTTTATTCCTATGTGAT283 rhabdomyosarcoma/ mir-135_2 hypothetical 1559 hypotheticalTGATCTTGCTCTAACACTTGG 1157 miRNA 170 miRNA-170 glutamate 174hypothetical TGACAAGTATGTTTTATCGTT 1158 receptor, miRNA-171 ionotropic,AMPA 2/hypothetical miRNA-171 hypothetical 179 hypotheticalTAGGAGTTTGATATGACATAT 1163 miRNA 176 miRNA-176 hypothetical 1560hypothetical TGAAAGGCACTTTGTCCAATT 1166 miRNA 179 miRNA-179 hypothetical1409 hypothetical TCACCTGCTCTGGAAGTAGTT 1167 miRNA 181 miRNA-181mir-181c 1410 mir-181c AACATTCAACCTGTCGGTGAGT 290 mir-100_1 1561 mir-100AACCCGTAGATCCGAACTTGTG 275 mir-103_1 950 mir-103 AGCAGCATTGTACAGGGCTATGA225 mir-107 1562 mir-107 AGCAGCATTGTACAGGGCTATCA 229 mir-19a 1563mir-19a TGTGCAAATCTATGCAAAACTGA 268 mir-19b_1 1414 mir-19b* (MichaelAGTTTTGCAGGTTTGCATCCAGC 1179 et al) mir-19b_1 1414 mir-19bTGTGCAAATCCATGCAAAACTGA 241 mir-92_1 1564 miR-92 (RFAM-M. mu.)TATTGCACTTGTCCCGGCCTG 1182 mir-92_1 1564 mir-92 TATTGCACTTGTCCCGGCCTGT216 mir-98 1565 mir-98 TGAGGTAGTAAGTTGTATTGTT 257 mir-104 1566 miR-104TCAACATCAGTCTGATAAGCTA 335 (Mourelatos) (Mourelatos) mir-27 1567 miR-27TTCACAGTGGCTAAGTTCC 1186 (Mourelatos) (Mourelatos) mir-27 1567 miR-27a(RFAM-M. mu.) TTCACAGTGGCTAAGTTCCGC 1187 (Mourelatos) mir-27 1567miR-27a (RFAM- TTCACAGTGGCTAAGTTCCGCC 1188 (Mourelatos) Human) mir-311568 miR-31 (RFAM-M. mu.) AGGCAAGATGCTGGCATAGCTG 1197 mir-31 1568 miR-31(Tuachl) GGCAAGATGCTGGCATAGCTG 1198 mir-32 1569 miR-32 (Tuachl)TATTGCACATTACTAAGTTGC 1199 mir_186 1570 miR-186 CAAAGAATTCTCCTTTTGGGCTT1208 mir_191 1571 mir-191 CAACGGAATCCCAAAAGCAGCT 1210 mir_191 1422mir-191_Ruvkun CAACGGAATCCCAAAAGCAGCTGT 1211 mir_195 1572 miR-195TAGCAGCACAGAAATATTGGC 1216 mir_193 1573 miR-193 AACTGGCCTACAAAGTCCCAG1217 mir_208 1574 miR-208 ATAAGACGAGCAAAAAGCTTGT 1222 mir_139 1427miR-139 TCTACAGTGCACGTGTCT 1223 mir-200b 1428 miR-200a (RFAM-CTCTAATACTGCCTGGTAATGATG 1224 Human) mir-200b 1428 miR-200b (MichaelTAATACTGCCTGGTAATGATGA 1225 et al) mir-200b 1428 miR-200bTAATACTGCCTGGTAATGATGAC 1226 mir-200a 1429 miR-200aTAACACTGTCTGGTAACGATG 1227 mir-200a 1429 miR-200a (RFAM-M. mu.)TAACACTGTCTGGTAACGATGT 1228 mir-227* 1430 mir-226* (Kosik)ACTGCCCCAGGTGCTGCTGG 1231 (Kosik)/mir-226* (Kosik) mir-227* 1430mir-324-3p_Ruvkun CCACTGCCCCAGGTGCTGCTGG 1232 (Kosik)/mir-226* (Kosik)mir-227* 1430 mir-227* (Kosik) CGCATCCCCTAGGGCATTGGTGT 1233(Kosik)/mir-226* (Kosik) mir-244* (Kosik) 1431 mir-244* (Kosik)TCCAGCATCAGTGATTTTGTTGA 1234 mir-224* (Kosik) 1432 mir-224* (Kosik)GCACATTACACGGTCGACCTCT 1235 mir-248* (Kosik) 1433 mir-248* (Kosik)TCTCACACAGAAATCGCACCCGTC 1236 mir-138_3 1575 mir-138 AGCTGGTGTTGTGAATC256 mir-138_3 1575 mir-138_Ruvkun AGCTGGTGTTGTGAATCAGGCCG 1127mir-181b_2 1576 mir-181b AACATTCATTGCTGTCGGTGGGTT 260 mir-134 (Sanger)1289 miR-134 (RFAM- TGTGACTGGTTGACCAGAGGG 1240 Human) mir-146 (Sanger)1577 miR-146 (RFAM- TGAGAACTGAATTCCATGGGTT 1241 Human) mir-30e 1578miR-30e (RFAM-M. mu.) TGTAAACATCCTTGACTGGA 1243 (RFAM/mmu) mir-30e 1578miR-97 (Michael TGTAAACATCCTTGACTGGAAG 1244 (RFAM/mmu) et al) mir-2991440 miR-299 (RFAM-M. mu.) TGGTTTACCGTCCCACATACAT 1246 (RFAM/mmu)mir-34a 1579 mir-34c (RFAM) AGGCAGTGTAGTTAGCTGATTG 1250 (RFAM/mmu)mir-34a 1579 miR-34a (RFAM-M. mu.) AGGCAGTGTAGTTAGCTGATTGC 1251(RFAM/mmu) mir-135b (Ruvkun) 1580 mir-135b (Ruvkun)TATGGCTTTTCATTCCTATGTG 1254 mir-331 (Ruvkun) 1442 mir-331 (Ruvkun)GCCCCTGGGCCTATCCTAGAA 1258 mir-187 1443 miR-187 (RFAM-TCGTGTCTTGTGTTGCAGCCG 1270 Human) mir-187 1443 mir-187TCGTGTCTTGTGTTGCAGCCGG 276 collagen, type I, 1581 hypotheticalAGACATGTTCAGCTTTGTGGA 1063 alpha 1/ miRNA-144 hypothetical miRNA-144DiGeorge syndrome 1582 hypothetical TGTGATTTCCAATAATTGAGG 1123 criticalregion miRNA-088 gene 8/ hypothetical miRNA-088 hypothetical miR- 1583miR-190 TGATATGTTTGATATATTAGGT 1075 13/miR-190 hypothetical 1584hypothetical TAAGACTTGCAGTGATGTTTA 1091 miRNA 039 miRNA-039 hypothetical1585 hypothetical TACCAGTTGTTTTCTCTGTGA 1093 miRNA 041 miRNA-041hypothetical 47 hypothetical TTCCACTCTGTTTATCTGACA 1095 miRNA 044miRNA-044 hypothetical 86 hypothetical TTACATGGGGAAGCTATCATA 1119 miRNA083 miRNA-083 hypothetical 1586 hypothetical TGACAGTTTATTGGCTTTATC 1133miRNA 107 miRNA-107 mir-10a 1587 mir-10a (Tuschl) TACCCTGTAGATCCGAATTTGT1139 mir-10a 1587 mir-10a TACCCTGTAGATCCGAATTTGTG 267 mir-130b 1588mir-130b CAGTGCAATGATGAAAGGGC 273 mir-130b 1588 mir-266* (Kosik)CAGTGCAATGATGAAAGGGCAT 1140 hypothetical 1589 hypotheticalAGACAAACATGCTACTCTCAC 1164 miRNA-177_1 miRNA-177 mir_185 1590 miR-185TGGAGAGAAAGGCAGTTC 1218 mir_194_2 1591 miR-194 TGTAACAGCAACTCCATGTGGA1221 mir-150 (Sanger) 1592 miR-150 (RFAM- TCTCCCAACCCTTGTACCAGTG 1242Human) mir-301 1593 miR-301 (RFAM-M. mu.) CAGTGCAATAGTATTGTCAAAGC 1247(RFAM/mmu) mir-301 1593 mir-301 _Ruvkun CAGTGCAATAGTATTGTCAAAGCAT 1248(RFAM/mmu) mir_320 1594 miR-320 AAAAGCTGGGTTGAGAGGGCGAA 1252 mir_200c(RFAM) 1595 mir-200c (RFAM) AATACTGCCGGGTAATGATGGA 1259 miR-322 1596miR-322 AAACATGAAGCGCTGCAACA 1489 miR-341 1457 miR-341TCGATCGGTCGGTCGGTCAGT 1494 miR-344 1597 miR-344 TGATCTAGCCAAAGCCTGACCGT1610 miR-350 1598 miR-350 TTCACAAAGCCCATACACTTTCAC 1491 miR-351 1599miR-351 TCCCTGAGGAGCCCTTTGAGCCTG 1493 miR-290 1600 miR-290CTCAAACTATGGGGGCACTTTTT 1492 miR-291 1601 miR-291AAAGTGCTTCCACTTTGTGTGCC 1481 miR-291 1601 miR-291 CATCAAAGTGGAGGCCCTCTCT1482 miR-292 1602 miR-292 AAGTGCCGCCAGGTTTTGAGTGT 1483 miR-292 1602miR-292 ACTCAAACTGGGGGCTCTTTTG 1484 miR-298 1603 miR-298GGCAGAGGAGGGCTGTTCTTCC 1495 miR-300 1604 miR-300 TATGCAAGGGCAAGCTCTCTTC1488 miR-333 1605 miR-333 GTGGTGTGCTAGTTACTTTT 1611 miR-336 1606 miR-336TCACCCTTCCATATCTAGTCT 1612 miR-349 1607 miR-349 CAGCCCTGCTGTCTTAACCTCT1613

A list of Drosophila pri-miRNAs and the mature miRNAs predicted toderive from them is shown in Table 63. “Pri-miRNA name” indicates thegene name for each of the pri-miRNAs, and “pri-miRNA sequence” indicatesthe sequence of the predicted primary miRNA transcript. Also given intable 63 are the name and sequence of the mature miRNA derived from thepri-miRNA. The sequences are written in the 5′ to 3′ direction and arerepresented in the DNA form. It is understood that a person havingordinary skill in the art would be able to convert the sequence of thetargets to their RNA form by simply replacing the thymidine (T) withuracil (U) in the sequence.

TABLE 63 Drosophila pri-miRNA sequences and the corresponding maturemiRNAs Pri- SEQ SEQ miRNA ID Mature miRNA ID name Pri-miRNA sequence NOname Mature miRNA sequence NO mir-14 GGAGCGAGACGGGGACTCACT 1614 miR-14TCAGTCTTTTTCTCTCTCCTA 1616 GTGCTTATTAAATAGTCAGTC TTTTTCTCTCTCCTATACAAATTGCGGGC mir- AATGATTTGACTACGAAACCG 1615 mir-BantamGTGAGATCATTTTGAAAGCTG 1617 bantam GTTTTCGATTTGGTTTGACTGTTTTTCATACAAGTGAGATCA TTTTGAAAGCTGATTTTGTCA ATGAATA

Oligomeric compounds targeting or mimicking pri-miRNAs, pre-miRNAs, ormiRNAs were given internal numerical identifiers (ISIS Numbers) and areshown in Tables 64, 65, and 66 respectively. The sequences are writtenin the 5′ to 3′ direction and are represented in the DNA form. It isunderstood that a person having ordinary skill in the art would be ableto convert the sequence of the targets to their RNA form by simplyreplacing the thymidine (T) with uracil (U) in the sequence.

Table 64 describes a series of oligomeric compounds designed andsynthesized to target different regions of pri-miRNAs. These oligomericcompounds can be analyzed for their effect on miRNA, pre-miRNA orpri-miRNA levels by quantitative real-time PCR, or they can be used inother assays to investigate the role of miRNAs or miRNA downstreamtargets. In Table 64, “Pri-miRNA” indicates the particular pri-miRNAwhich contains the miRNA that the oligomeric compound was designed totarget. All compounds listed in Table 64 have phosphorothioateinternucleoside linkages. In some embodiments, chimeric oligonucleotides(“gapmers”) are composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five nucleotide “wings,” wherein the wings are composedof 2′-methoxyethoxy (2′-MOE) nucleotides. These chimeric compounds areindicated in the “Chemistry” column as “5-10-5 MOE gapmer.” In someembodiments, oligomeric compound consist of 2′-MOE ribonucleotidesthroughout, and these are indicated by “uniform MOE.”

TABLE 64 Phosphorothioate oligomeric compounds targeting pri-miRNAs SEQID ISIS # NO sequence chemistry Pri-miRNA 338615 442AGAACAGCATGACGTAACCT uniform MOE mir-140, Human 338616 443GCCCATCTGTGGCTTCACAG uniform MOE mir-30a, Human 338617 444GAAGTCCGAGGCAGTAGGCA uniform MOE mir-30a, Human 338618 445CTTCCTTACTATTGCTCACA uniform MOE mir-34, Human 338619 446GCTAGATACAAAGATGGAAA uniform MOE mir-29b-1, Human 338620 447CTAGACAATCACTATTTAAA uniform MOE mir-29b-2, Human 338621 448GCAGCGCAGCTGGTCTCCCC uniform MOE mir-29b-2, Human 338622 449TAATATATATTTCACTACGC uniform MOE mir-16-3, Human 338623 450TGCTGTATCCCTGTCACACT uniform MOE mir-16-3, Human 338624 451CAATTGCGCTACAGAACTGT uniform MOE mir-203, Human 338625 452TCGATTTAGTTATCTAAAAA uniform MOE mir-7-1, Human 338626 453CTGTAGAGGCATGGCCTGTG uniform MOE mir-7-1, Human 338627 454TGACTATACGGATACCACAC uniform MOE mir-10b, Human 338628 455GGAACAAGGCCAATTATTGC uniform MOE mir-128a, Human 338629 456AGAAATGTAAACCTCTCAGA uniform MOE mir-128a, Human 338630 457AGCTGTGAGGGAGAGAGAGA uniform MOE mir-153-1, Human 338631 458CTGGAGTGAGAATACTAGCT uniform MOE mir-153-1, Human 338632 459ACTGGGCTCATATTACTAGC uniform MOE mir-153-2, Human 338633 460TTGGATTAAATAACAACCTA uniform MOE hypothetical miR- 13/miR-190, Human338634 461 CCCGGAGACAGGGCAAGACA uniform MOE hypothetical miR-13/miR-190, Human 338635 462 AAAGCGGAAACCAATCACTG uniform MOE chromosome9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338636 463GTCCCCATCTCACCTTCTCT uniform MOE chromosome 9 ORF3 containing mir-23b,mir-24-2 and mir-27b, Human 338637 464 TCAGAGCGGAGAGACACAAG uniform MOEmir-96, Human 338638 465 TAGATGCACATATCACTACC uniform MOE miR-17/mir-91,Human 338639 466 CTTGGCTTCCCGAGGCAGCT uniform MOE miR-17/mir-91, Human338640 467 AGTTTGAAGTGTCACAGCGC uniform MOE mir-123/mir-126, Human338641 468 GCGTTTTCGATGCGGTGCCG uniform MOE mir-123/mir-126, Human338642 469 GAGACGCGGGGGCGGGGCGC uniform MOE mir-132, Human 338643 470TACCTCCAGTTCCCACAGTA uniform MOE mir-132, Human 338644 471TGTGTTTTCTGACTCAGTCA uniform MOE mir-108-1, Human 338645 472AGAGCACCTGAGAGCAGCGC uniform MOE chromosome 9 ORF3 containing mir-23b,mir-24-2 and mir-27b, Human 338646 473 TCTTAAGTCACAAATCAGCA uniform MOEchromosome 9 ORF3 containing mir-23b, mir-24-2 and mir-27b, Human 338647474 TCTCCACAGCGGGCAATGTC uniform MOE let-7i, Human 338648 475GGCGCGCTGTCCGGGCGGGG uniform MOE mir-212, Human 338649 476ACTGAGGGCGGCCCGGGCAG uniform MOE mir-212, Human 338650 477GTCCTCTTGCCCAAGCAACA uniform MOE hypothetical miRNA-023, Human 338651478 GAAGACCAATACACTCATAC uniform MOE mir-131-2/miR-9, Human 338652 479CCGAGGGGCAACATCACTGC uniform MOE let-7b, Human 338653 480TCCATAGCTTAGCAGGTCCA uniform MOE mir-1d-1, Human 338654 481TTTGATAGTTTAGACACAAA uniform MOE mir-122a, Human 338655 482GGGAAGGATTGCCTAGCAGT uniform MOE mir-122a, Human 338656 483AGCTTTAGCTGGGTCAGGAC uniform MOE mir-22, Human 338657 484TACCATACAGAAACACAGCA uniform MOE mir-92-1, Human 338658 485TCACAATCCCCACCAAACTC uniform MOE mir-92-1, Human 338659 486TCACTCCTAAAGGTTCAAGT uniform MOE hypothetical miRNA-30, Human 338660 487CACCCTCCAGTGCTGTTAGT uniform MOE mir-142, Human 338661 488CTGACTGAGACTGTTCACAG uniform MOE mir-183, Human 338662 489CCTTTAGGGGTTGCCACACC uniform MOE glutamate receptor, ionotrophic, AMPA3/ hypothetical miRNA-033, Human 338663 490 ACAGGTGAGCGGATGTTCTG uniformMOE mir-214, Human 338665 492 AGAGGGGAGACGAGAGCACT uniform MOEmir-192-1, Human 338666 493 TCACGTGGAGAGGAGTTAAA uniform MOEhypothetical miRNA-039, Human 338667 494 AGTGCTAATACTTCTTTCAT uniformMOE hypothetical miRNA-040, Human 338668 495 ACCTGTGTAACAGCCGTGTAuniform MOE hypothetical miRNA-041, Human 338669 496TTATCGGAACTTCACAGAGA uniform MOE hypothetical miRNA-041, Human 338670497 TCCCATAGCAGGGCAGAGCC uniform MOE let-7a-3, Human 338671 498GGCACTTCATTGCTGCTGCC uniform MOE hypothetical miRNA-043, Human 338672499 GGAGCCTTGCGCTCAGCATT uniform MOE hypothetical miRNA-043, Human338673 500 ATGGTAATTTCATTTCAGGC uniform MOE hypothetical miRNA-044,Human 338674 501 GATTGCACATCCACACTGTC uniform MOE hypotheticalmiRNA-044, Human 338675 502 GCTGGCCTGATAGCCCTTCT uniform MOE mir-181a,Human 338676 503 GTTTTTTCAAATCCCAAACT uniform MOE mir-181a, Human 338677504 CCCAGTGGTGGGTGTGACCC uniform MOE let-7a-1, Human 338678 505CTGGTTGGGTATGAGACAGA uniform MOE mir-205, Human 338679 506TTGATCCATATGCAACAAGG uniform MOE mir-103-1, Human 338680 507GCCATTGGGACCTGCACAGC uniform MOE miR-26a-1, Human 338681 508ATGGGTACCACCAGAACATG uniform MOE mir-33a, Human 338682 509AGTTCAAAACTCAATCCCAA uniform MOE mir-196-2, Human 338683 510GCCCTCGACGAAAACCGACT uniform MOE mir-196-2, Human 338684 511TTGAACTCCATGCCACAAGG uniform MOE mir-107, Human 338685 512AGGCCTATTCCTGTAGCAAA uniform MOE mir-106, Human 338686 513GTAGATCTCAAAAAGCTACC uniform MOE mir-106, Human 338687 514CTGAACAGGGTAAAATCACT uniform MOE let-7f-1, Human 338688 515AGCAAGTCTACTCCTCAGGG uniform MOE let-7f-1, Human 338689 516AATGGAGCCAAGGTGCTGCC uniform MOE hypothetical miRNA-055, Human 338690517 TAGACAAAAACAGACTCTGA uniform MOE mir-29c, Human 338691 518GCTAGTGACAGGTGCAGACA uniform MOE mir-130a, Human 338692 519GGGCCTATCCAAAGTGACAG uniform MOE hypothetical miRNA-058, Human 338693520 TACCTCTGCAGTATTCTACA uniform MOE hypothetical miRNA-058, Human338694 521 TTTACTCATACCTCGCAACC uniform MOE mir-218-1, Human 338695 522AATTGTATGACATTAAATCA uniform MOE mir-124a-2, Human 338696 523CTTCAAGTGCAGCCGTAGGC uniform MOE mir-124a-2, Human 338697 524TGCCATGAGATTCAACAGTC uniform MOE mir-21, Human 338698 525ACATTGCTATCATAAGAGCT uniform MOE mir-16-1, Human 338699 526TAATTTTAGAATCTTAACGC uniform MOE mir-16-1, Human 338700 527AGTGTCTCATCGCAAACTTA uniform MOE mir-144, Human 338701 528TGTTGCCTAACGAACACAGA uniform MOE mir-221, Human 338702 529GCTGATTACGAAAGACAGGA uniform MOE mir-222, Human 338703 530GCTTAGCTGTGTCTTACAGC uniform MOE mir-30d, Human 338704 531GAGGATGTCTGTGAATAGCC uniform MOE mir-30d, Human 338705 532CCACATATACATATATACGC uniform MOE mir-19b-2, Human 338706 533AGGAAGCACACATTATCACA uniform MOE mir-19b-2, Human 338707 534GACCTGCTACTCACTCTCGT uniform MOE mir-128b, Human 338708 535GGTTGGCCGCAGACTCGTAC uniform MOE hypothetical miRNA 069/mir-219-2, Human338709 536 GATGTCACTGAGGAAATCAC uniform MOE hypothetical miRNA-070,Human 338710 537 TCAGTTGGAGGCAAAAACCC uniform MOE LOC 114614/hypothetical miRNA-071, Human 338711 538 GGTAGTGCAGCGCAGCTGGT uniformMOE mir-29b-2, Human 338712 539 CCGGCTATTGAGTTATGTAC uniform MOEmir-129-2, Human 338713 540 ACCTCTCAGGAAGACGGACT uniform MOE mir-133b,Human 338714 541 GAGCATGCAACACTCTGTGC uniform MOE hypotheticalmiRNA-075, Human 338715 542 CCTCCTTGTGGGCAAAATCC uniform MOE let-7d,Human 338716 543 CGCATCTTGACTGTAGCATG uniform MOE mir-15b, Human 338717544 TCTAAGGGGTCACAGAAGGT uniform MOE mir-29a-1, Human 338718 545GAAAATTATATTGACTCTGA uniform MOE mir-29a-1, Human 338719 546GGTTCCTAATTAAACAACCC uniform MOE hypothetical miRNA-079, Human 338720547 CCGAGGGTCTAACCCAGCCC uniform MOE mir-199b, Human 338721 548GACTACTGTTGAGAGGAACA uniform MOE mir-129-1, Human 338722 549TCTCCTTGGGTGTCCTCCTC uniform MOE let-7e, Human 338723 550TGCTGACTGCTCGCCCTTGC uniform MOE hypothetical miRNA-083, Human 338724551 ACTCCCAGGGTGTAACTCTA uniform MOE let7c-1, Human 338725 552CATGAAGAAAGACTGTAGCC uniform MOE mir-204, Human 338726 553GACAAGGTGGGAGCGAGTGG uniform MOE mir-145, Human 338727 554TGCTCAGCCAGCCCCATTCT uniform MOE mir-124a-1, Human 338728 555GCTTTTAGAACCACTGCCTC uniform MOE DiGeorge syndrome critical region gene8/ hypothetical miRNA-088, Human 338729 556 GGAGTAGATGATGGTTAGCC uniformMOE mir-213/mir-181a, Human 338730 557 ACTGATTCAAGAGCTTTGTA uniform MOEhypothetical miRNA-090, Human 338731 558 GTAGATAACTAAACACTACC uniformMOE mir-20, Human 338732 559 AATCCATTGAAGAGGCGATT uniform MOEmir-133a-1, Human 338733 560 GGTAAGAGGATGCGCTGCTC uniform MOE mir-138-2,Human 338734 561 GGCCTAATATCCCTACCCCA uniform MOE mir-98, Human 338735562 GTGTTCAGAAACCCAGGCCC uniform MOE mir-196-1, Human 338736 563TCCAGGATGCAAAAGCACGA uniform MOE mir-125b-1, Human 338737 564TACAACGGCATTGTCCTGAA uniform MOE mir-199a-2, Human 338738 565TTTCAGGCTCACCTCCCCAG uniform MOE hypothetical miRNA-099, Human 338739566 AAAAATAATCTCTGCACAGG uniform MOE mir-181b, Human 338740 567AGAATGAGTTGACATACCAA uniform MOE hypothetical miRNA-101, Human 338741568 GCTTCACAATTAGACCATCC uniform MOE mir-141, Human 338742 569AGACTCCACACCACTCATAC uniform MOE mir-131-1/miR-9, Human 338743 570ATCCATTGGACAGTCGATTT uniform MOE mir-133a-2, Human 338744 571GGCGGGCGGCTCTGAGGCGG uniform MOE hypothetical miRNA-105, Human 338745572 CTCTTTAGGCCAGATCCTCA uniform MOE hypothetical miRNA-105, Human338746 573 TAATGGTATGTGTGGTGATA uniform MOE hypothetical miRNA-107,Human 338747 574 ATTACTAAGTTGTTAGCTGT uniform MOE miR-1d-2, Human 338748575 GATGCTAATCTACTTCACTA uniform MOE mir-18, Human 338749 576TCAGCATGGTGCCCTCGCCC uniform MOE mir-220, Human 338750 577TCCGCGGGGGCGGGGAGGCT uniform MOE hypothetical miRNA-111, Human 338751578 AGACCACAGCCACTCTAATC uniform MOE mir-7-3, Human 338752 579TCCGTTTCCATCGTTCCACC uniform MOE mir-218-2, Human 338753 580GCCAGTGTACACAAACCAAC uniform MOE mir-24-2, Human 338754 581AAGGCTTTTTGCTCAAGGGC uniform MOE chromosome 9 ORF3 containing mir-23b,mir-24-2 and mir-27b, Human 338755 582 TTGACCTGAATGCTACAAGG uniform MOEmir-103-2, Human 338756 583 TGCCCTGCTCAGAGCCCTAG uniform MOE mir-211,Human 338757 584 TCAATGTGATGGCACCACCA uniform MOE mir-101-3, Human338758 585 ACCTCCCAGCCAATCCATGT uniform MOE mir-30b, Human 338759 586TCCTGGATGATATCTACCTC uniform MOE hypothetical miRNA-120, Human 338760587 TCTCCCTTGATGTAATTCTA uniform MOE let-7a-4, Human 338761 588AGAGCGGAGTGTTTATGTCA uniform MOE mir-10a, Human 338762 589TCATTCATTTGAAGGAAATA uniform MOE mir-19a, Human 338763 590TCCAAGATGGGGTATGACCC uniform MOE let-7f-2, Human 338764 591TTTTTAAACACACATTCGCG uniform MOE mir-15a-1, Human 338765 592AGATGTGTTTCCATTCCACT uniform MOE mir-108-2, Human 338766 593CCCCCTGCCGCTGGTACTCT uniform MOE mir-137, Human 338767 594CGGCCGGAGCCATAGACTCG uniform MOE mir-219-1, Human 338768 595CTTTCAGAGAGCCACAGCCT uniform MOE mir-148b, Human 338769 596GCTTCCCAGCGGCCTATAGT uniform MOE mir-130b, Human 338770 597CAGCAGAATATCACACAGCT uniform MOE mir-19b-1, Human 338771 598TACAATTTGGGAGTCCTGAA uniform MOE mir-199b, Human 338772 599GCCTCCTTCATATATTCTCA uniform MOE mir-204, Human 338773 600CCCCATCTTAGCATCTAAGG uniform MOE mir-145, Human 338774 601TTGTATGGACATTTAAATCA uniform MOE mir-124a-1, Human 338775 602TTTGATTTTAATTCCAAACT uniform MOE mir-213/mir-181a, Human 338776 603CAAACGGTAAGATTTGCAGA uniform MOE hypothetical miRNA-090, Human 338777604 GGATTTAAACGGTAAACATC uniform MOE mir-125b-1, Human 338778 605CTCTAGCTCCCTCACCAGTG uniform MOE hypothetical miRNA-099, Human 338779606 GCTTGTCCACACAGTTCAAC uniform MOE mir-181b, Human 338780 607GCATTGTATGTTCATATGGG uniform MOE miR-1d-2, Human 338781 608TGTCGTAGTACATCAGAACA uniform MOE mir-7-3, Human 338782 609AGCCAGTGTGTAAAATGAGA uniform MOE chromosome 9 ORF3 containing mir-23b,mir-24-2 and mir-27b, Human 338783 610 TTCAGATATACAGCATCGGT uniform MOEmir-101-3, Human 338784 611 TGACCACAAAATTCCTTACA uniform MOE mir-10a,Human 338785 612 ACAACTACATTCTTCTTGTA uniform MOE mir-19a, Human 338786613 TGCACCTTTTCAAAATCCAC uniform MOE mir-15a-1, Human 338787 614AACGTAATCCGTATTATCCA uniform MOE mir-137, Human 338788 615CGTGAGGGCTAGGAAATTGC uniform MOE mir-216, Human 338789 616GCAACAGGCCTCAATATCTT uniform MOE mir-100-1, Human 338790 617ACGAGGGGTCAGAGCAGCGC uniform MOE mir-187, Human 338791 618GGCAGACGAAAGGCTGACAG uniform MOE hypothetical miRNA-137, Human 338792619 CTGCACCATGTTCGGCTCCC uniform MOE hypothetical miRNA-138, Human338793 620 GGGGCCCTCAGGGCTGGGGC uniform MOE mir-124a-3, Human 338794 621CCGGTCCACTCTGTATCCAG uniform MOE mir-7-2, Human 338795 622GCTGGGAAAGAGAGGGCAGA uniform MOE hypothetical miRNA-142, Human 338796623 TCAGATTGCCAACATTGTGA uniform MOE hypothetical miRNA-143, Human338797 624 CTGGGGAGGGGGTTAGCGTC uniform MOE collagen, type I, alpha1/hypothetical miRNA- 144, Human 338798 625 TGGGTCTGGGGCAGCGCAGT uniformMOE mir-210, Human 338799 626 TTGAAGTAGCACAGTCATAC uniform MOE mir-215,Human 338800 627 TCTACCACATGGAGTGTCCA uniform MOE mir-223, Human 338801628 AGTGCCGCTGCCGCGCCGTG uniform MOE mir-131-3/miR-9, Human 338802 629ACACATTGAGAGCCTCCTGA uniform MOE mir-199a-1, Human 338803 630GTCGCTCAGTGCTCTCTAGG uniform MOE mir-30c-1, Human 338804 631AGGCTCCTCTGATGGAAGGT uniform MOE mir-101-1, Human 338805 632GCTGTGACTTCTGATATTAT uniform MOE hypothetical miRNA-153, Human 338806633 GACATCATGTGATTTGCTCA uniform MOE hypothetical miRNA-154, Human338807 634 CACCCCAAGGCTGCAGGGCA uniform MOE mir-26b, Human 338808 635TGTCAAGCCTGGTACCACCA uniform MOE hypothetical miRNA-156, Human 338809636 CTGCTCCAGAGCCCGAGTCG uniform MOE mir-152, Human 338810 637ACCCTCCGCTGGCTGTCCCC uniform MOE mir-135-1, Human 338811 638TAGAGTGAATTTATCTTGGT uniform MOE non-coding RNA in rhabdomyosarcoma/mir-135-2, Human 338812 639 TGGTGACTGATTCTTATCCA uniform MOE mir-217, Human338813 640 CAATATGATTGGATAGAGGA uniform MOE hypothetical miRNA-161,Human 338814 641 TTTAAACACACATTCGCGCC uniform MOE mir-15a-1, Human338815 642 ACCGGGTGGTATCATAGACC uniform MOE let-7g, Human 338816 643TGCATACCTGTTCAGTTGGA uniform MOE hypothetical miRNA-164, Human 338817644 GCCCGCCTCTCTCGGCCCCC uniform MOE sterol regulatory element-bindingprotein-1/mir-33b, Human 338818 645 TCGCCCCCTCCCAGGCCTCT uniform MOEhypothetical miRNA-166, Human 338819 646 ACAACTGTAGAGTATGGTCA uniformMOE mir-16-1, Human 338820 647 GCTGACCATCAGTACTTTCC uniform MOEhypothetical miRNA 168- 1/similar to ribosomal protein L5, Human 338821648 TTATAGAACAGCCTCCAGTG uniform MOE forkhead box P2/hypothetical miRNA-169, Human 338822 649 TTCAGGCACTAGCAGTGGGT uniform MOE hypotheticalmiRNA-170, Human 338823 650 AGTACTGCGAGGTTAACCGC uniform MOE glutamatereceptor, ionotropic, AMPA 2/ hypothetical miRNA-171, Human 338824 651GGACCTTTAAGATGCAAAGT uniform MOE hypothetical miRNA-172, Human 338825652 TTCATATTATCCACCCAGGT uniform MOE hypothetical miRNA-173, Human338826 653 CGGATCCTGTTACCTCACCA uniform MOE mir-182, Human 338827 654TGGTGCCTGCCACATCTTTG uniform MOE hypothetical miRNA-175, Human 338828655 TGGGAGGCTGAATCAAGGAC uniform MOE hypothetical miRNA-176, Human338829 656 TGACAACCAGGAAGCTTGTG uniform MOE hypothetical miRNA-177- 1,Human 338830 657 GCCAGGCAGCGAGCTTTTGA uniform MOE hypotheticalmiRNA-178, Human 338831 658 CAGCCTGCCACCGCCGCTTT uniform MOEhypothetical miRNA-179, Human 338832 659 CTGCCCCCGTGGACCGAACA uniformMOE cezanne 2/hypothetical miRNA-180, Human 338833 660TCGTGCACCTGAGGAGTCTG uniform MOE hypothetical miRNA-181, Human 338834661 CAAACGTGCTGTCTTCCTCC uniform MOE mir-148a, Human 338835 662AAGGACTCAGCAGTGTTTCA uniform MOE tight junction protein 1 (zonaoccludens 1)/ hypothetical miRNA-183, Human 338836 663TCCTCGGTGGCAGAGCTCAG uniform MOE mir-23a, Human 338837 664AGACAATGAGTACACAGTTC uniform MOE hypothetical miRNA-185, Human 338838665 CTGCAAGCACTGGTTCCCAT uniform MOE hypothetical miRNA-177-2/hypothetical miRNA 186, Human 338839 666 TTGCCTGAGCTGCCCAAACT uniformMOE mir-181c, Human 338840 667 TCCATCACACTGTCCTATGA uniform MOEhypothetical miRNA-188, Human 338841 668 GAGGGATTGTATGAACATCT uniformMOE mir-216, Human 338842 669 GCTTGTGCGGACTAATACCA uniform MOEmir-100-1, Human 338843 670 GCAGGCTAAAAGAAATAAGC uniform MOEhypothetical miRNA-138, Human 338844 671 ATTGTATAGACATTAAATCA uniformMOE mir-124a-3, Human 338845 672 GTTGAGCGCAGTAAGACAAC uniform MOEmir-7-2, Human 338846 673 AGATGTTTCTGGCCTGCGAG uniform MOE hypotheticalmiRNA-142, Human 338847 674 GACAAACTCAGCTATATTGT uniform MOE mir-215,Human 338848 675 ACGGCTCTGTGGCACTCATA uniform MOE mir-131-3/miR-9, Human338849 676 GCTTTCTTACTTTCCACAGC uniform MOE mir-30c-1, Human 338850 677TACCTTTAGAATAGACAGCA uniform MOE mir-101-1, Human 338851 678AGGCTGGACAGCACACAACC uniform MOE mir-26b, Human 338852 679AGCAGGAGCCTTATCTCTCC uniform MOE hypothetical miRNA-156, Human 338853680 ATGAGTGAGCAGTAGAATCA uniform MOE mir-135-1, Human 338854 681TGAGACTTTATTACTATCAC uniform MOE non-coding RNA in rhabdomyosarcoma/mir-135-2, Human 338855 682 TACTTTACTCCAAGGTTTTA uniform MOE mir-15a-1,Human 338856 683 GCACCCGCCTCACACACGTG uniform MOE sterol regulatoryelement-binding protein-1/mir-33b, Human 338857 684 TTCCCGACCTGCCTTTACCTuniform MOE hypothetical miRNA-166, Human 338858 685TCCTGTAATTATAGGCTAGC uniform MOE forkhead box P2/hypothetical miRNA-169, Human 338859 686 GGATCATATCAATAATACCA uniform MOE hypotheticalmiRNA-172, Human 338860 687 TGCTGAGACACACAATATGT uniform MOEhypothetical miRNA-176, Human 338861 688 TGTTTGTCTCCAAGAAACGT uniformMOE hypothetical miRNA-177- 1, Human 338862 689 TGTCATGGACAGGATGAATAuniform MOE hypothetical miRNA-179, Human 338863 690TCTATCATACTCAGAGTCGG uniform MOE mir-148a, Human 338864 691TTGTGACAGGAAGCAAATCC uniform MOE mir-23a, Human 338865 692CATCAGAGTCACCAACCCCA uniform MOE hypothetical miRNA-185, Human 338866693 CAAGAGATGTCTCGTTTTGC uniform MOE hypothetical miRNA-177-2/hypothetical miRNA 186, Human 340342 937 GACTGTTGAATCTCATGGCA uniformMOE miR-104 (Mourelatos), Human 340344 1656 GCATGAGCAGCCACCACAGG uniformMOE miR-105 (Mourelatos), Human 340346 1626 ACGACTTGGTGTGGACCCTG uniformMOE miR-27 (Mourelatos), Human 340347 849 TACTTTATATAGAACACAAG uniformMOE mir-92-2/miR-92 (Mourelatos), Human 340349 1632 AGGTTGGGTAATCACACTACuniform MOE miR-93 (Mourelatos), Human 340351 1621 AATGTAACGCATTTCAATTCuniform MOE miR-95 (Mourelatos), Human 340353 1694 TGTGCGGTCCACTTCACCACuniform MOE miR-99 (Mourelatos), Human 340355 1671 GTCCAGCAATTGCCCAAGTCuniform MOE miR-25, Human 340357 1662 GGAAAGTCAGAAAGGTAACT uniform MOEmiR-28, Human 340359 1635 CAGGTTCCCAGTTCAACAGC uniform MOE miR-31, Human340361 1636 CATTGAGGCCGTGACAACAT uniform MOE miR-32, Human 340363 1656GCATGAGCAGCCACCACAGG 5-10-5 MOE miR-105 (Mourelatos), gapmer Human340364 1626 ACGACTTGGTGTGGACCCTG 5-10-5 MOE miR-27 (Mourelatos), gapmerHuman 340366 1632 AGGTTGGGTAATCACACTAC 5-10-5 MOE miR-93 (Mourelatos),gapmer Human 340367 1621 AATGTAACGCATTTCAATTC 5-10-5 MOE miR-95(Mourelatos), gapmer Human 340368 1694 TGTGCGGTCCACTTCACCAC 5-10-5 MOEmiR-99 (Mourelatos), gapmer Human 340369 1671 GTCCAGCAATTGCCCAAGTC5-10-5 MOE miR-25, Human gapmer 340370 1662 GGAAAGTCAGAAAGGTAACT 5-10-5MOE miR-28, Human gapmer 340371 1635 CAGGTTCCCAGTTCAACAGC 5-10-5 MOEmiR-31, Human gapmer 340372 1636 CATTGAGGCCGTGACAACAT 5-10-5 MOE miR-32,Human gapmer 341817 1630 AGCCACCTTGAGCTCACAGC uniform MOE miR-30c-2,Human 341818 1695 TGTGTGCGGCGAAGGCCCCG uniform MOE miR-99b, Human 3418191657 GCCAGGCTCCCAAGAACCTC uniform MOE MiR-125a, Human 341820 1653GATGTTACTAAAATACCTCA uniform MOE MiR-125b-2, Human 341822 1679TCCGATGATCTTTCTGAATC uniform MOE miR-127, Human 341825 1646CTTAAAATAAAACCAGAAAG uniform MOE miR-186, Human 341826 1618AAAATCACAGGAACCTATCT uniform MOE miR-198, Human 341827 1688TGGAATGCTCTGGAGACAAC uniform MOE miR-191, Human 341828 1677TCCATAGCAAAGTAATCCAT uniform MOE miR-206, Human 341829 1668GGTAGCACGGAGAGGACCAC uniform MOE miR-94, Human 341830 1624ACACTTACAGTCACAAAGCT uniform MOE miR-184, Human 341831 1654GCAGACTCGCTTCCCTGTGC uniform MOE miR-195, Human 341832 1684TGATCCGACACCCTCATCTC uniform MOE miR-193, Human 341833 1641CCTGGGGAGGGGACCATCAG uniform MOE miR-185, Human 341834 1676TCAGAAAGCTCACCCTCCAC uniform MOE miR-188, Human 341835 1648GAGCTCTTACCTCCCACTGC uniform MOE miR-197, Human 341836 1686TGGAAATTGGTACACAGTCC uniform MOE miR-194-1, Human 341837 1642CGTGAGCATCAGGTATAACC uniform MOE miR-208, Human 341838 1687TGGAACCAGTGGGCACTTCC uniform MOE miR-194-2, Human 341839 1638CCAGCCTCCGAGCCACACTG uniform MOE miR-139, Human 341840 1628AGACCTGACTCCATCCAATG uniform MOE miR-200b, Human 341841 1629AGAGTCAAGCTGGGAAATCC uniform MOE miR-200a, Human 341843 1630AGCCACCTTGAGCTCACAGC 5-10-5 MOE miR-30c-2, Human gapmer 341844 1695TGTGTGCGGCGAAGGCCCCG 5-10-5 MOE miR-99b, Human gapmer 341845 1657GCCAGGCTCCCAAGAACCTC 5-10-5 MOE MiR-125a, Human gapmer 341846 1653GATGTTACTAAAATACCTCA 5-10-5 MOE MiR-125b-2, Human gapmer 341848 1679TCCGATGATCTTTCTGAATC 5-10-5 MOE miR-127, Human gapmer 341851 1646CTTAAAATAAAACCAGAAAG 5-10-5 MOE miR-186, Human gapmer 341852 1618AAAATCACAGGAACCTATCT 5-10-5 MOE miR-198, Human gapmer 341853 1688TGGAATGCTCTGGAGACAAC 5-10-5 MOE miR-191, Human gapmer 341854 1677TCCATAGCAAAGTAATCCAT 5-10-5 MOE miR-206, Human gapmer 341855 1668GGTAGCACGGAGAGGACCAC 5-10-5 MOE miR-94, Human gapmer 341856 1624ACACTTACAGTCACAAAGCT 5-10-5 MOE miR-184, Human gapmer 341857 1654GCAGACTCGCTTCCCTGTGC 5-10-5 MOE miR-195, Human gapmer 341858 1684TGATCCGACACCCTCATCTC 5-10-5 MOE miR-193, Human gapmer 341859 1641CCTGGGGAGGGGACCATCAG 5-10-5 MOE miR-185, Human gapmer 341860 1676TCAGAAAGCTCACCCTCCAC 5-10-5 MOE miR-188, Human gapmer 341861 1648GAGCTCTTACCTCCCACTGC 5-10-5 MOE miR-197, Human gapmer 341862 1686TGGAAATTGGTACACAGTCC 5-10-5 MOE miR-194-1, Human gapmer 341863 1642CGTGAGCATCAGGTATAACC 5-10-5 MOE miR-208, Human gapmer 341864 1687TGGAACCAGTGGGCACTTCC 5-10-5 MOE miR-194-2, Human gapmer 341865 1638CCAGCCTCCGAGCCACACTG 5-10-5 MOE miR-139, Human gapmer 341866 1628AGACCTGACTCCATCCAATG 5-10-5 MOE miR-200b, Human gapmer 341867 1629AGAGTCAAGCTGGGAAATCC 5-10-5 MOE miR-200a, Human gapmer 344731 1619AACGGTTTATGACAAACATT uniform MOE mir-240* (Kosik), Human 344732 1665GGGCTGTATGCACTTTCTCC uniform MOE mir-232* (Kosik), Human 344733 1667GGGTCTCCAGCTTTACACCA uniform MOE mir-227* (Kosik)/mir- 226* (Kosik),Human 344734 1649 GAGTCGCCTGAGTCATCACT uniform MOE mir-244* (Kosik),Human 344735 1658 GCCATAAATAAAGCGAACGC uniform MOE mir-224* (Kosik),Human 344736 1678 TCCATTAACCATGTCCCTCA uniform MOE mir-248* (Kosik),Human 344737 1619 AACGGTTTATGACAAACATT 5-10-5 MOE mir-240* (Kosik),Human gapmer 344738 1665 GGGCTGTATGCACTTTCTCC 5-10-5 MOE mir-232*(Kosik), Human gapmer 344739 1667 GGGTCTCCAGCTTTACACCA 5-10-5 MOEmir-227* (Kosik)/mir- gapmer 226* (Kosik), Human 344740 1649GAGTCGCCTGAGTCATCACT 5-10-5 MOE mir-244* (Kosik), Human gapmer 3447411658 GCCATAAATAAAGCGAACGC 5-10-5 MOE mir-224* (Kosik), Human gapmer344742 1678 TCCATTAACCATGTCCCTCA 5-10-5 MOE mir-248* (Kosik), Humangapmer 346787 1689 TGGCTTCCATAGTCTGGTGT uniform MOE miR-147 (Sanger),Human 346788 1623 ACAATGCACAATCATCTACT uniform MOE miR-224 (Sanger),Human 346789 1669 GGTGAACACAGTGCATGCCC uniform MOE miR-134 (Sanger),Human 346790 1682 TCTGACACTGACACAACCCA uniform MOE miR-146 (Sanger),Human 346791 1631 AGGGTCTGAGCCCAGCACTG uniform MOE miR-150 (Sanger),Human 346792 1637 CCAAGAGACGTTTCATTTTG uniform MOE hypotheticalmiRNA-177- 3, Human 346793 1683 TCTGATTGGCAACGGCCTGA uniform MOEmir-138-3, Human 346794 1627 ACTGTCCATCTTAGTTCAGA uniform MOE mir-138-4,Human 346795 1634 AGTTGATTCAGACTCAAACC uniform MOE mir-181b-2, Human346796 1655 GCATAAGCAGCCACCACAGG uniform MOE miR-105-2, Human 3467971691 TGTATGATATCTACCTCAGG uniform MOE hypothetical miRNA-120- 2, Human346798 1689 TGGCTTCCATAGTCTGGTGT 5-10-5 MOE miR-147 (Sanger), Humangapmer 346799 1623 ACAATGCACAATCATCTACT 5-10-5 MOE miR-224 (Sanger),Human gapmer 346800 1669 GGTGAACACAGTGCATGCCC 5-10-5 MOE miR-134(Sanger), Human gapmer 346801 1682 TCTGACACTGACACAACCCA 5-10-5 MOEmiR-146 (Sanger), Human gapmer 346802 1631 AGGGTCTGAGCCCAGCACTG 5-10-5MOE miR-150 (Sanger), Human gapmer 346803 1637 CCAAGAGACGTTTCATTTTG5-10-5 MOE hypothetical miRNA-177- gapmer 3, Human 346804 1683TCTGATTGGCAACGGCCTGA 5-10-5 MOE mir-138-3, Human gapmer 346805 1627ACTGTCCATCTTAGTTCAGA 5-10-5 MOE mir-138-4, Human gapmer 346806 1634AGTTGATTCAGACTCAAACC 5-10-5 MOE mir-181b-2, Human gapmer 346807 1655GCATAAGCAGCCACCACAGG 5-10-5 MOE miR-105-2, Human gapmer 346808 1691TGTATGATATCTACCTCAGG 5-10-5 MOE hypothetical miRNA-120- gapmer 2, Human348225 1620 AAGAGAAGGCGGAGGGGAGC 5-10-5 MOE miR-320, Human gapmer 3482261643 CTCGAACCCACAATCCCTGG 5-10-5 MOE miR-321-1, Human gapmer 354006 1650GAGTTTGGGACAGCAATCAC 5-10-5 MOE mir-135b (Ruvkun), gapmer Human 3540071633 AGTAGGGGATGAGACATACT 5-10-5 MOE mir-151* (Ruvkun), gapmer Human354008 1639 CCCACAAACGACATATGACA 5-10-5 MOE mir-340 (Ruvkun), Humangapmer 354009 1664 GGCCTGGTTTGATCTGGGAT 5-10-5 MOE mir-331 (Ruvkun),Human gapmer 354010 1647 GAGACTCCCAACCGCACCCA 5-10-5 MOE miR-200c(RFAM-Human) gapmer 354011 1700 TTGTAACCACCACAGTACAA 5-10-5 MOE miR-34b(RFAM-Human) gapmer 354012 1663 GGAGGACAGGGAGAGCGGCC 5-10-5 MOEmir-339-1 (RFAM-Human) gapmer 354013 1675 TCACAGGCAGGCACACGTGA 5-10-5MOE mir-339-1 (RFAM-Human) gapmer 354014 1698 TTCAGAGCTACAGCATCGGT5-10-5 MOE mir-101-3, Mouse gapmer 354015 1670 GTAGAACTCAAAAAGCTACC5-10-5 MOE mir-106, Mouse gapmer 354016 1673 TAGATGCACACATCACTACC 5-10-5MOE miR-17/mir-91, Mouse gapmer 354017 1690 TGTACAATTTGGGAGTCCTG 5-10-5MOE mir-199b, Human gapmer 354018 1644 CTCTTTAGACCAGATCCACA 5-10-5 MOEhypothetical miRNA-105, gapmer Mouse 354019 1640 CCTCACTCAGAGGCCTAGGC5-10-5 MOE mir-211, Mouse gapmer 354020 1666 GGGGATTAAGTCTTATCCAG 5-10-5MOE mir-217, Mouse gapmer 354021 1622 ACAATGCACAAACCATCTAC 5-10-5 MOEmiR-224 (Sanger), Mouse gapmer 354022 1693 TGTCATATCATATCAGAACA 5-10-5MOE mir-7-3, Mouse gapmer 354023 1672 TAGATGACGACACACTACCT 5-10-5 MOEmir-20, Rat gapmer 354024 1692 TGTCACAAACACTTACTGGA 5-10-5 MOE mir-325(Ruvkun), Human gapmer 354025 1625 ACGAATTATGTCACAAACAC 5-10-5 MOEmir-325 (Ruvkun), Mouse gapmer 354026 1651 GATCTGAGCACCACCCGCCT 5-10-5MOE mir-326 (Ruvkun), Human gapmer 354027 1652 GATCTGAGCATAACCCGCCT5-10-5 MOE mir-326 (Ruvkun), Mouse gapmer 354028 1697TGTTTCGTCCTCATTAAAGA 5-10-5 MOE mir-329-1 (Ruvkun), gapmer Human 3540291699 TTCTCATCAAAGAAACAGAG 5-10-5 MOE mir-329-1 (Ruvkun), gapmer Mouse354030 1696 TGTTTCGTCCTCAATAAAGA 5-10-5 MOE mir-329-2 (Ruvkun), gapmerHuman 354031 1681 TCGGTTGATCTTGCAGAGCC 5-10-5 MOE mir-330 (Ruvkun),Human gapmer 354032 1685 TGCTCGTTGGATCTTGAAGA 5-10-5 MOE mir-330(Ruvkun), Mouse gapmer 354033 1661 GCTGGATAACTGTGCATCAA 5-10-5 MOEmir-337 (Ruvkun), Human gapmer 354034 1645 CTGAATGGCTGTGCAATCAA 5-10-5MOE mir-337 (Ruvkun), Mouse gapmer 354035 1659 GCCCACCAGCCATCACGAGC5-10-5 MOE mir-345 (Ruvkun), Human gapmer 354036 1660GCCCAGTAGCCACCACAAGC 5-10-5 MOE mir-345 (Ruvkun), Mouse gapmer 3540371680 TCCTTCAGAGCAACAGAGAG 5-10-5 MOE mir-346 (Ruvkun), Human gapmer354038 1674 TAGTAGGGAGGAGACATACT 5-10-5 MOE mir-151* (Ruvkun), gapmerMouse 354039 1701 TTGTCAGCACCGCACTACAA 5-10-5 MOE miR-34b (RFAM-Mouse)gapmer

In accordance with the present invention, a further series of oligomericcompounds were designed and synthesized to target different regions ofmiRNAs. These oligomeric compounds can be analyzed for their effect onmiRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, orthey can be used in other assays to investigate the role of miRNAs ormiRNA downstream targets. The compounds are shown in Table 65, where“pri-miRNA” indicates the particular pri-miRNA which contains the miRNAthat the oligomeric compound was designed to target. Oligomericcompounds having phosphorothioate internucleoside linkages are indicatedby “PS” in the “Chemistry” column of Table 65, whereas compounds havingphosphodiester internucleoside linkages are indicated by “PO.” In someembodiments, chimeric oligonucleotides (“gapmers”) are composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by nucleotide “wings” twoto ten nucleotides in length. The wings are composed of 2′-methoxyethoxy(2′-MOE) ribonucleotides. In some embodiments, chimeric oligonucleotidesare of the “open end” type wherein the “gap” segment is located ateither the 3′ or the 5′ terminus of the oligomeric compound. Chimericoligonucleotides of this type are also known in the art and areindicated in Table 65 as “hemimers.” For example, “PO/6MOE-10deoxyhemimer,” describes a chimeric oligomeric compound consisting of six2′-MOE ribonucleotides at the 5′-terminus, followed by tendeoxyribonucleotides on the 3′-terminal end, with a phosphodiesterbackbone throughout the hemimer.

TABLE 65 Oligomeric compounds targeting miRNAs SEQ ID ISIS # NO sequenceChemistry Pri-miRNA 340343 1780 ACAGGAGTCTGAGCATTTGA PS/MOE miR-105(Mourelatos) 340345 1882 GGAACTTAGCCACTGTGAA PS/MOE miR-27 (Mourelatos)340350 855 TGCTCAATAAATACCCGTTGAA PS/MOE miR-95 (Mourelatos) 340352 1821CACAAGATCGGATCTACGGGTT PS/MOE miR-99 (Mourelatos) 340354 1903TCAGACCGAGACAAGTGCAATG PS/MOE miR-25 (Tuschl) 340356 1853CTCAATAGACTGTGAGCTCCTT PS/MOE miR-28 (Tuschl) 340358 1825CAGCTATGCCAGCATCTTGCC PS/MOE miR-31 (Tuschl) 340360 1865GCAACTTAGTAATGTGCAATA PS/MOE miR-32 (Tuschl) 340924 298ACAAATTCGGTTCTACAGGGTA PS/MOE 5-10-7 mir-10b gapmer 340925 307GTGGTAATCCCTGGCAATGTGAT PS/MOE 5-10-8 mir-23b gapmer 340928 322ACTCACCGACAGCGTTGAATGTT PS/MOE 5-10-8 mir-181a gapmer 340929 331AACCGATTTCAAATGGTGCTAG PS/MOE 5-10-7 mir-29c gapmer 340930 342GCAAGCCCAGACCGCAAAAAG PS/MOE 5-10-6 mir-129 gapmer 340931 346AACCGATTTCAGATGGTGCTAG PS/MOE 5-10-7 mir-29a gapmer 340932 349AACCATACAACCTACTACCTCA PS/MOE 5-10-7 let-7c gapmer 340933 352GGTACAATCAACGGTCGATGGT PS/MOE 5-10-7 mir-213 gapmer 340934 356AACAATACAACTTACTACCTCA PS/MOE 5-10-7 mir-98 gapmer 340935 373GCCCTTTCATCATTGCACTG PS/MOE 5-10-5 mir-130b gapmer 340936 385ACTGTACAAACTACTACCTCA PS/MOE 5-10-6 let-7g gapmer 341785 854GGAGTGAAGACACGGAGCCAGA PS/MOE miR-149 341786 1845 CGCAAGGTCGGTTCTACGGGTGPS/MOE miR-99b 341787 852 CACAGGTTAAAGGGTCTCAGGGA PS/MOE miR-125a 341788853 AGCCAAGCTCAGACGGATCCGA PS/MOE miR-127 341789 1909TCCATCATCAAAACAAATGGAGT PS/MOE miR-136 341790 1843CGAAGGCAACACGGATAACCTA PS/MOE miR-154 341791 1880GCTTCCAGTCGAGGATGTTTACA PS/MOE miR-30a-s 341792 1911TCCGTGGTTCTACCCTGTGGTA PS/MOE miR-140-as 341793 1836CCATAAAGTAGGAAACACTACA PS/MOE miR-142-as 341794 1761AACAGGTAGTCTGAACACTGGG PS/MOE miR-199-s 341795 1762AACCAATGTGCAGACTACTGTA PS/MOE miR-199-as 341796 1904TCATACAGCTAGATAACCAAAGA PS/MOE miR-9 341797 1773 ACAAGTGCCTTCACTGCAGTPS/MOE miR-17 341798 1871 GCATTATTACTCACGGTACGA PS/MOE miR-126a 3417991787 ACCTAATATATCAAACATATCA PS/MOE miR-190 341800 1766AAGCCCAAAAGGAGAATTCTTTG PS/MOE miR-186 341801 1839 CCTATCTCCCCTCTGGACCPS/MOE miR-198a 341802 1806 AGCTGCTTTTGGGATTCCGTTG PS/MOE miR-191c341803 760 CCACACACTTCCTTACATTCCA PS/MOE miR-206d 341804 761ATCTGCACTGTCAGCACTTT PS/MOE miR-94 341805 762 ACCCTTATCAGTTCTCCGTCCAPS/MOE miR-184 341806 763 GCCAATATTTCTGTGCTGCTA PS/MOE miR-195 341807764 CTGGGACTTTGTAGGCCAGTT PS/MOE miR-193 341808 1861 GAACTGCCTTTCTCTCCAPS/MOE miR-185 341809 1786 ACCCTCCACCATGCAAGGGATG PS/MOE miR-188 3418101879 GCTGGGTGGAGAAGGTGGTGAA PS/MOE miR-197a 341811 1906TCCACATGGAGTTGCTGTTACA PS/MOE miR-194 341812 1771 ACAAGCTTTTTGCTCGTCTTATPS/MOE miR-208 341814 1887 GTCATCATTACCAGGCAGTATTA PS/MOE miR-200b341815 1831 CATCGTTACCAGACAGTGTTA PS/MOE miR-200a 342946 1897TAGGAGAGAGAAAAAGACTGA PS/MOE miR-14 342947 1827 CAGCTTTCAAAATGATCTCACPS/MOE miR-Bantam 343875 321 AACTATACAACCTACTACCTCA PO/MOE let-7a 3442671769 ACAAATTCGGATCTACAGGGTA PS/MOE miR-10 (Tuschl) 344268 1774ACACAAATTCGGTTCTACAGGG PS/MOE miR-10b (Tuschl) 344269 1890TAACCGATTTCAAATGGTGCTA PS/MOE miR-29c (Tuschl) 344270 1867GCACGAACAGCACTTTG PS/MOE miR-93 (Tuschl) 344271 1770ACAAGATCGGATCTACGGGT PS/MOE miR-99a (Tuschl) 344272 1816CAAACACCATTGTCACACTCCA PS/MOE miR-122a, b (Tuschl) 344273 1920TGTCAATTCATAGGTCAG PS/MOE miR-192 (Tuschl) 344274 1832CCAACAACATGAAACTACCTA PS/MOE miR-196 (Tuschl) 344275 1912TCTAGTGGTCCTAAACATTTCA PS/MOE miR-203 (Tuschl) 344276 1828CAGGCATAGGATGACAAAGGGAA PS/MOE miR-204 (Tuschl) 344277 1767AATACATACTTCTTTACATTCCA PS/MOE miR-1d (Tuschl) 344278 1769ACAAATTCGGATCTACAGGGTA PS/MOE 5-10-7 miR-10 (Tuschl) gapmer 344279 1774ACACAAATTCGGTTCTACAGGG PS/MOE 5-10-7 miR-10b gapmer (Tuschl) 344280 1890TAACCGATTTCAAATGGTGCTA PS/MOE 5-10-7 miR-29c gapmer (Tuschl) 344281 1867GCACGAACAGCACTTTG PS/MOE 5-10-2 miR-93 (Tuschl) gapmer 344282 1770ACAAGATCGGATCTACGGGT PS/MOE 5-10-5 miR-99a gapmer (Tuschl) 344283 1816CAAACACCATTGTCACACTCCA PS/MOE 5-10-7 miR-122a, b gapmer (Tuschl) 3442841920 TGTCAATTCATAGGTCAG PS/MOE 5-10-3 miR-192 gapmer (Tuschl) 3442851832 CCAACAACATGAAACTACCTA PS/MOE 5-10-6 miR-196 gapmer (Tuschl) 3442861912 TCTAGTGGTCCTAAACATTTCA PS/MOE 5-10-7 miR-203 gapmer (Tuschl) 3442871828 CAGGCATAGGATGACAAAGGGAA PS/MOE 5-10-8 miR-204 gapmer (Tuschl)344288 1767 AATACATACTTCTTTACATTCCA PS/MOE 5-10-8 miR-1d (Tuschl) gapmer344336 1918 TGGCATTCACCGCGTGCCTTA PS/MOE mir-124a (Kosik) 344337 1754AAAGAGACCGGTTCACTGTGA PS/MOE mir-128 (Kosik) 344338 1812ATGCCCTTTTAACATTGCACTG PS/MOE mir-130 (Kosik) 344339 1854CTCACCGACAGCGTTGAATGTT PS/MOE mir-178 (Kosik) 344340 1921TGTCCGTGGTTCTACCCTGTGGTA PS/MOE mir-239* (Kosik) 344341 1823CACATGGTTAGATCAAGCACAA PS/MOE mir-253* (Kosik) 344342 1814ATGCTTTTTGGGGTAAGGGCTT PS/MOE mir-129as/mir- 258* (Kosik) 344343 1811ATGCCCTTTCATCATTGCACTG PS/MOE mir-266* (Kosik) 344344 1918TGGCATTCACCGCGTGCCTTA PS/MOE 5-10-6 mir-124a gapmer (Kosik) 344345 1754AAAGAGACCGGTTCACTGTGA PS/MOE 5-10-6 mir-128 (Kosik) gapmer 344346 1812ATGCCCTTTTAACATTGCACTG PS/MOE 5-10-7 mir-130 (Kosik) gapmer 344347 1854CTCACCGACAGCGTTGAATGTT PS/MOE 5-10-7 mir-178 (Kosik) gapmer 344348 1921TGTCCGTGGTTCTACCCTGTGGTA PS/MOE 5-10-9 mir-239* gapmer (Kosik) 3443491823 CACATGGTTAGATCAAGCACAA PS/MOE 5-10-7 mir-253* gapmer (Kosik) 3443501814 ATGCTTTTTGGGGTAAGGGCTT PS/MOE 5-10-7 mir-129as/mir- gapmer 258*(Kosik) 344351 1811 ATGCCCTTTCATCATTGCACTG PS/MOE 5-10-7 mir-266* gapmer(Kosik) 344611 1785 ACATTTTTCGTTATTGCTCTTGA PS/MOE mir-240* (Kosik)344612 1790 ACGGAAGGGCAGAGAGGGCCAG PS/MOE mir-232* (Kosik) 344613 1775ACACCAATGCCCTAGGGGATGCG PS/MOE mir-227* (Kosik) 344614 1834CCAGCAGCACCTGGGGCAGT PS/MOE mir-226* (Kosik) 344615 1900TCAACAAAATCACTGATGCTGGA PS/MOE mir-244* (Kosik) 344616 1800AGAGGTCGACCGTGTAATGTGC PS/MOE mir-224* (Kosik) 344617 1862GACGGGTGCGATTTCTGTGTGAGA PS/MOE mir-248* (Kosik) 344618 1785ACATTTTTCGTTATTGCTCTTGA PS/MOE 5-10-8 mir-240* gapmer (Kosik) 3446191790 ACGGAAGGGCAGAGAGGGCCAG PS/MOE 5-10-7 mir-232* gapmer (Kosik) 3446201775 ACACCAATGCCCTAGGGGATGCG PS/MOE 5-10-8 mir-227* gapmer (Kosik)344621 1834 CCAGCAGCACCTGGGGCAGT PS/MOE 5-10-5 mir-226* gapmer (Kosik)344622 1900 TCAACAAAATCACTGATGCTGGA PS/MOE 5-10-8 mir-244* gapmer(Kosik) 344623 1800 AGAGGTCGACCGTGTAATGTGC PS/MOE 5-10-7 mir-224* gapmer(Kosik) 344624 1862 GACGGGTGCGATTTCTGTGTGAGA PS/MOE 5-10-9 mir-248*gapmer (Kosik) 345344 291 CTACCATAGGGTAAAACCACT PS/MOE 5-10-6 mir-140gapmer 345345 292 GCTGCAAACATCCGACTGAAAG PS/MOE 5-10-7 mir-30a gapmer345346 293 ACAACCAGCTAAGACACTGCCA PS/MOE 5-10-7 mir-34 gapmer 345347 294AACACTGATTTCAAATGGTGCTA PS/MOE 5-10-8 mir-29b gapmer 345348 295CGCCAATATTTACGTGCTGCTA PS/MOE 5-10-7 mir-16 gapmer 345350 297AACAAAATCACTAGTCTTCCA PS/MOE 5-10-6 mir-7 gapmer 345351 299AAAAGAGACCGGTTCACTGTGA PS/MOE 5-10-7 mir-128a gapmer 345352 300TCACTTTTGTGACTATGCAA PS/MOE 5-10-5 mir-153 gapmer 345353 301CAGAACTTAGCCACTGTGAA PS/MOE 5-10-5 mir-27b gapmer 345354 302GCAAAAATGTGCTAGTGCCAAA PS/MOE 5-10-7 mir-96 gapmer 345355 303ACTACCTGCACTGTAAGCACTTTG PS/MOE 5-10-9 mir-17as/mir-91 gapmer 345356 304CGCGTACCAAAAGTAATAATG PS/MOE 5-10-6 mir-123/mir- gapmer 126as 345357 305GCGACCATGGCTGTAGACTGTTA PS/MOE 5-10-8 mir-132 gapmer 345358 306AATGCCCCTAAAAATCCTTAT PS/MOE 5-10-6 mir-108 gapmer 345359 308AGCACAAACTACTACCTCA PS/MOE 5-10-4 let-7i gapmer 345360 309GGCCGTGACTGGAGACTGTTA PS/MOE 5-10-6 mir-212 gapmer 345361 311AACCACACAACCTACTACCTCA PS/MOE 5-10-7 let-7b gapmer 345362 312ATACATACTTCTTTACATTCCA PS/MOE 5-10-7 mir-1d gapmer 345363 313ACAAACACCATTGTCACACTCCA PS/MOE 5-10-8 mir-122a gapmer 345364 314ACAGTTCTTCAACTGGCAGCTT PS/MOE 5-10-7 mir-22 gapmer 345365 315ACAGGCCGGGACAAGTGCAATA PS/MOE 5-10-7 mir-92 gapmer 345366 316GTAGTGCTTTCTACTTTATG PS/MOE 5-10-5 mir-142 gapmer 345367 317CAGTGAATTCTACCAGTGCCATA PS/MOE 5-10-8 mir-183 gapmer 345368 318CTGCCTGTCTGTGCCTGCTGT PS/MOE 5-10-6 mir-214 gapmer 345369 320GGCTGTCAATTCATAGGTCAG PS/MOE 5-10-6 mir-192 gapmer 345370 321AACTATACAACCTACTACCTCA PS/MOE 5-10-7 let-7a gapmer 345371 323CAGACTCCGGTGGAATGAAGGA PS/MOE 5-10-7 mir-205 gapmer 345372 324TCATAGCCCTGTACAATGCTGCT PS/MOE 5-10-8 mir-103 gapmer 345373 325AGCCTATCCTGGATTACTTGAA PS/MOE 5-10-7 mir-26a gapmer 345374 326CAATGCAACTACAATGCAC PS/MOE 5-10-4 mir-33a gapmer 345375 327CCCAACAACATGAAACTACCTA PS/MOE 5-10-7 mir-196 gapmer 345376 328TGATAGCCCTGTACAATGCTGCT PS/MOE 5-10-8 mir-107 gapmer 345377 329GCTACCTGCACTGTAAGCACTTTT PS/MOE 5-10-9 mir-106 gapmer 345378 330AACTATACAATCTACTACCTCA PS/MOE 5-10-7 let-7f gapmer 345379 332GCCCTTTTAACATTGCACTG PS/MOE 5-10-5 mir-130a gapmer 345380 333ACATGGTTAGATCAAGCACAA PS/MOE 5-10-6 mir-218 gapmer 345381 334TGGCATTCACCGCGTGCCTTAA PS/MOE 5-10-7 mir-124a gapmer 345382 335TCAACATCAGTCTGATAAGCTA PS/MOE 5-10-7 mir-21 gapmer 345383 336CTAGTACATCATCTATACTGTA PS/MOE 5-10-7 mir-144 gapmer 345384 337GAAACCCAGCAGACAATGTAGCT PS/MOE 5-10-8 mir-221 gapmer 345385 338GAGACCCAGTAGCCAGATGTAGCT PS/MOE 5-10-9 mir-222 gapmer 345386 339CTTCCAGTCGGGGATGTTTACA PS/MOE 5-10-7 mir-30d gapmer 345387 340TCAGTTTTGCATGGATTTGCACA PS/MOE 5-10-8 mir-19b gapmer 345388 341GAAAGAGACCGGTTCACTGTGA PS/MOE 5-10-7 mir-128b gapmer 345389 343TAGCTGGTTGAAGGGGACCAA PS/MOE 5-10-6 mir-133b gapmer 345390 344ACTATGCAACCTACTACCTCT PS/MOE 5-10-6 let-7d gapmer 345391 345TGTAAACCATGATGTGCTGCTA PS/MOE 5-10-7 mir-15b gapmer 345392 347GAACAGATAGTCTAAACACTGGG PS/MOE 5-10-8 mir-199b gapmer 345393 348ACTATACAACCTCCTACCTCA PS/MOE 5-10-6 let-7e gapmer 345394 350AGGCATAGGATGACAAAGGGAA PS/MOE 5-10-7 mir-204 gapmer 345395 351AAGGGATTCCTGGGAAAACTGGAC PS/MOE 5-10-9 mir-145 gapmer 345396 353CTACCTGCACTATAAGCACTTTA PS/MOE 5-10-8 mir-20 gapmer 345397 354ACAGCTGGTTGAAGGGGACCAA PS/MOE 5-10-7 mir-133a gapmer 345398 355GATTCACAACACCAGCT PS/MOE 5-10-2 mir-138 gapmer 345399 357TCACAAGTTAGGGTCTCAGGGA PS/MOE 5-10-7 mir-125b gapmer 345400 358GAACAGGTAGTCTGAACACTGGG PS/MOE 5-10-8 mir-199a gapmer 345401 359AACCCACCGACAGCAATGAATGTT PS/MOE 5-10-9 mir-181b gapmer 345402 360CCATCTTTACCAGACAGTGTT PS/MOE 5-10-6 mir-141 gapmer 345403 361TATCTGCACTAGATGCACCTTA PS/MOE 5-10-7 mir-18 gapmer 345404 362AAAGTGTCAGATACGGTGTGG PS/MOE 5-10-6 mir-220 gapmer 345405 363CTGTTCCTGCTGAACTGAGCCA PS/MOE 5-10-7 mir-24 gapmer 345406 364AGGCGAAGGATGACAAAGGGAA PS/MOE 5-10-7 mir-211 gapmer 345407 365TCAGTTATCACAGTACTGTA PS/MOE 5-10-5 mir-101 gapmer 345408 366GCTGAGTGTAGGATGTTTACA PS/MOE 5-10-6 mir-30b gapmer 345409 367CACAAATTCGGATCTACAGGGTA PS/MOE 5-10-8 mir-10a gapmer 345410 368TCAGTTTTGCATAGATTTGCACA PS/MOE 5-10-8 mir-19a gapmer 345411 369CACAAACCATTATGTGCTGCTA PS/MOE 5-10-7 mir-15a gapmer 345412 370CTACGCGTATTCTTAAGCAATA PS/MOE 5-10-7 mir-137 gapmer 345413 371AGAATTGCGTTTGGACAATCA PS/MOE 5-10-6 mir-219 gapmer 345414 372ACAAAGTTCTGTGATGCACTGA PS/MOE 5-10-7 mir-148b gapmer 345415 374CACAGTTGCCAGCTGAGATTA PS/MOE 5-10-6 mir-216 gapmer 345416 375CACAAGTTCGGATCTACGGGTT PS/MOE 5-10-7 mir-100 gapmer 345417 376CCGGCTGCAACACAAGACACGA PS/MOE 5-10-7 mir-187 gapmer 345418 377CAGCCGCTGTCACACGCACAG PS/MOE 5-10-6 mir-210 gapmer 345419 378GTCTGTCAATTCATAGGTCAT PS/MOE 5-10-6 mir-215 gapmer 345420 379GGGGTATTTGACAAACTGACA PS/MOE 5-10-6 mir-223 gapmer 345421 380GCTGAGAGTGTAGGATGTTTACA PS/MOE 5-10-8 mir-30c gapmer 345422 381AACCTATCCTGAATTACTTGAA PS/MOE 5-10-7 mir-26b gapmer 345423 382CCAAGTTCTGTCATGCACTGA PS/MOE 5-10-6 mir-152 gapmer 345424 383ATCACATAGGAATAAAAAGCCATA PS/MOE 5-10-9 mir-135 gapmer 345425 384ATCCAATCAGTTCCTGATGCAGTA PS/MOE 5-10-9 mir-217 gapmer 345426 386CAATGCAACAGCAATGCAC PS/MOE 5-10-4 mir-33b gapmer 345427 387TGTGAGTTCTACCATTGCCAAA PS/MOE 5-10-7 mir-182 gapmer 345428 388ACAAAGTTCTGTAGTGCACTGA PS/MOE 5-10-7 mir-148a gapmer 345429 389GGAAATCCCTGGCAATGTGAT PS/MOE 5-10-6 mir-23a gapmer 345430 390ACTCACCGACAGGTTGAATGTT PS/MOE 5-10-7 mir-181c gapmer 345431 391ACTGTAGGAATATGTTTGATA PS/MOE 5-10-6 hypothetical gapmer miRNA-013 345432392 ATTAAAAAGTCCTCTTGCCCA PS/MOE 5-10-6 hypothetical gapmer miRNA-023345433 393 GCTGCCGTATATGTGATGTCA PS/MOE 5-10-6 hypothetical gapmermiRNA-030 345434 394 GGTAGGTGGAATACTATAACA PS/MOE 5-10-6 hypotheticalgapmer miRNA-033 345435 395 TAAACATCACTGCAAGTCTTA PS/MOE 5-10-6hypothetical gapmer miRNA-039 345436 396 TTGTAAGCAGTTTTGTTGACA PS/MOE5-10-6 hypothetical gapmer miRNA-040 345437 397 TCACAGAGAAAACAACTGGTAPS/MOE 5-10-6 hypothetical gapmer miRNA-041 345438 398CCTCTCAAAGATTTCCTGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-043 345439399 TGTCAGATAAACAGAGTGGAA PS/MOE 5-10-6 hypothetical gapmer miRNA-044345440 400 GAGAATCAATAGGGCATGCAA PS/MOE 5-10-6 hypothetical gapmermiRNA-055 345441 401 AAGAACATTAAGCATCTGACA PS/MOE 5-10-6 hypotheticalgapmer miRNA-058 345442 402 AATCTCTGCAGGCAAATGTGA PS/MOE 5-10-6hypothetical gapmer miRNA-070 345443 403 AAACCCCTATCACGATTAGCA PS/MOE5-10-6 hypothetical gapmer miRNA-071 345444 404 GCCCCATTAATATTTTAACCAPS/MOE 5-10-6 hypothetical gapmer miRNA-075 345445 405CCCAATATCAAACATATCA PS/MOE 5-10-4 hypothetical gapmer miRNA-079 345446406 TATGATAGCTTCCCCATGTAA PS/MOE 5-10-6 hypothetical gapmer miRNA-083345447 407 CCTCAATTATTGGAAATCACA PS/MOE 5-10-6 hypothetical gapmermiRNA-088 345448 408 ATTGATGCGCCATTTGGCCTA PS/MOE 5-10-6 hypotheticalgapmer miRNA-090 345449 409 CTGTGACTTCTCTATCTGCCT PS/MOE 5-10-6hypothetical gapmer miRNA-099 345450 410 AAACTTGTTAATTGACTGTCA PS/MOE5-10-6 hypothetical gapmer miRNA-101 345451 411 AAAGAAGTATATGCATAGGAAPS/MOE 5-10-6 hypothetical gapmer miRNA-105 345452 412GATAAAGCCAATAAACTGTCA PS/MOE 5-10-6 hypothetical gapmer miRNA-107 345453413 TCCGAGTCGGAGGAGGAGGAA PS/MOE 5-10-6 hypothetical gapmer miRNA-111345454 414 ATCATTACTGGATTGCTGTAA PS/MOE 5-10-6 hypothetical gapmermiRNA-120 345455 415 CAAAAATTATCAGCCAGTTTA PS/MOE 5-10-6 hypotheticalgapmer miRNA-137 345456 416 AATCTCATTTTCATACTTGCA PS/MOE 5-10-6hypothetical gapmer miRNA-138 345457 417 AGAAGGTGGGGAGCAGCGTCA PS/MOE5-10-6 hypothetical gapmer miRNA-142 345458 418 CAAAATTGCAAGCAAATTGCAPS/MOE 5-10-6 hypothetical gapmer miRNA-143 345459 419TCCACAAAGCTGAACATGTCT PS/MOE 5-10-6 hypothetical gapmer miRNA-144 345460420 TATTATCAGCATCTGCTTGCA PS/MOE 5-10-6 hypothetical gapmer miRNA-153345461 421 AATAACACACATCCACTTTAA PS/MOE 5-10-6 hypothetical gapmermiRNA-154 345462 422 AAGAAGGAAGGAGGGAAAGCA PS/MOE 5-10-6 hypotheticalgapmer miRNA-156 345463 423 ATGACTACAAGTTTATGGCCA PS/MOE 5-10-6hypothetical gapmer miRNA-161 345464 424 CAAAACATAAAAATCCTTGCA PS/MOE5-10-6 hypothetical gapmer miRNA-164 345465 425 TTACAGGTGCTGCAACTGGAAPS/MOE 5-10-6 hypothetical gapmer miRNA-166 345466 426AGCAGGTGAAGGCACCTGGCT PS/MOE 5-10-6 hypothetical gapmer miRNA-168 345467427 TATGAAATGCCAGAGCTGCCA PS/MOE 5-10-6 hypothetical gapmer miRNA-169345468 428 CCAAGTGTTAGAGCAAGATCA PS/MOE 5-10-6 hypothetical gapmermiRNA-170 345469 429 AACGATAAAACATACTTGTCA PS/MOE 5-10-6 hypotheticalgapmer miRNA-171 345470 430 AGTAACTTCTTGCAGTTGGA PS/MOE 5-10-5hypothetical gapmer miRNA-172 345471 431 AGCCTCCTTCTTCTCGTACTA PS/MOE5-10-6 hypothetical gapmer miRNA-173 345472 432 ACCTCAGGTGGTTGAAGGAGAPS/MOE 5-10-6 hypothetical gapmer miRNA-175 345473 433ATATGTCATATCAAACTCCTA PS/MOE 5-10-6 hypothetical gapmer miRNA-176 345474434 GTGAGAGTAGCATGTTTGTCT PS/MOE 5-10-6 hypothetical gapmer miRNA-177345475 435 TGAAGGTTCGGAGATAGGCTA PS/MOE 5-10-6 hypothetical gapmermiRNA-178 345476 436 AATTGGACAAAGTGCCTTTCA PS/MOE 5-10-6 hypotheticalgapmer miRNA-179 345477 437 ACCGAACAAAGTCTGACAGGA PS/MOE 5-10-6hypothetical gapmer miRNA-180 345478 438 AACTACTTCCAGAGCAGGTGA PS/MOE5-10-6 hypothetical gapmer miRNA-181 345479 439 GTAAGCGCAGCTCCACAGGCTPS/MOE 5-10-6 hypothetical gapmer miRNA-183 345480 440GAGCTGCTCAGCTGGCCATCA PS/MOE 5-10-6 hypothetical gapmer miRNA-185 345481441 TACTTTTCATTCCCCTCACCA PS/MOE 5-10-6 hypothetical gapmer miRNA-188345482 236 TAGCTTATCAGACTGATGTTGA PS/MOE 5-10-7 miR-104 gapmer(Mourelatos) 345483 1780 ACAGGAGTCTGAGCATTTGA PS/MOE 5-10-5 miR-105gapmer (Mourelatos) 345484 1882 GGAACTTAGCCACTGTGAA PS/MOE 5-10-4 miR-27gapmer (Mourelatos) 345485 848 CTACCTGCACGAACAGCACTTT PS/MOE 5-10-7miR-93 gapmer (Mourelatos) 345486 855 TGCTCAATAAATACCCGTTGAA PS/MOE5-10-7 miR-95 gapmer (Mourelatos) 345487 1821 CACAAGATCGGATCTACGGGTTPS/MOE 5-10-7 miR-99 gapmer (Mourelatos) 345488 1903TCAGACCGAGACAAGTGCAATG PS/MOE 5-10-7 miR-25 (Tuschl) gapmer 345489 1853CTCAATAGACTGTGAGCTCCTT PS/MOE 5-10-7 miR-28 (Tuschl) gapmer 345490 1825CAGCTATGCCAGCATCTTGCC PS/MOE 5-10-6 miR-31 (Tuschl) gapmer 345491 1865GCAACTTAGTAATGTGCAATA PS/MOE 5-10-6 miR-32 (Tuschl) gapmer 345492 1897TAGGAGAGAGAAAAAGACTGA PS/MOE 5-10-6 miR-14 gapmer 345493 854GGAGTGAAGACACGGAGCCAGA PS/MOE 5-10-7 miR-149 gapmer 345494 1845CGCAAGGTCGGTTCTACGGGTG PS/MOE 5-10-7 miR-99b gapmer 345495 852CACAGGTTAAAGGGTCTCAGGGA PS/MOE 5-10-8 miR-125a gapmer 345496 853AGCCAAGCTCAGACGGATCCGA PS/MOE 5-10-7 miR-127 gapmer 345497 1909TCCATCATCAAAACAAATGGAGT PS/MOE 5-10-8 miR-136 gapmer 345498 1843CGAAGGCAACACGGATAACCTA PS/MOE 5-10-7 miR-154 gapmer 345499 1880GCTTCCAGTCGAGGATGTTTACA PS/MOE 5-10-8 miR-30a-s gapmer 345500 1911TCCGTGGTTCTACCCTGTGGTA PS/MOE 5-10-7 miR-140-as gapmer 345501 1836CCATAAAGTAGGAAACACTACA PS/MOE 5-10-7 miR-142-as gapmer 345502 1761AACAGGTAGTCTGAACACTGGG PS/MOE 5-10-7 miR-199-s gapmer 345503 1762AACCAATGTGCAGACTACTGTA PS/MOE 5-10-7 miR-199-as gapmer 345504 1904TCATACAGCTAGATAACCAAAGA PS/MOE 5-10-8 miR-9 gapmer 345505 1773ACAAGTGCCTTCACTGCAGT PS/MOE 5-10-5 miR-17 gapmer 345506 1871GCATTATTACTCACGGTACGA PS/MOE 5-10-6 miR-126a gapmer 345507 1787ACCTAATATATCAAACATATCA PS/MOE 5-10-7 miR-190 gapmer 345508 1766AAGCCCAAAAGGAGAATTCTTTG PS/MOE 5-10-8 miR-186 gapmer 345509 1839CCTATCTCCCCTCTGGACC PS/MOE 5-10-4 miR-198a gapmer 345510 1806AGCTGCTTTTGGGATTCCGTTG PS/MOE 5-10-7 miR-191c gapmer 345511 760CCACACACTTCCTTACATTCCA PS/MOE 5-10-7 miR-206d gapmer 345512 761ATCTGCACTGTCAGCACTTT PS/MOE 5-10-5 miR-94 gapmer 345513 762ACCCTTATCAGTTCTCCGTCCA PS/MOE 5-10-7 miR-184 gapmer 345514 763GCCAATATTTCTGTGCTGCTA PS/MOE 5-10-6 miR-195 gapmer 345515 764CTGGGACTTTGTAGGCCAGTT PS/MOE 5-10-6 miR-193 gapmer 345516 1861GAACTGCCTTTCTCTCCA PS/MOE 5-10-3 miR-185 gapmer 345517 1786ACCCTCCACCATGCAAGGGATG PS/MOE 5-10-7 miR-188 gapmer 345518 1879GCTGGGTGGAGAAGGTGGTGAA PS/MOE 5-10-7 miR-197a gapmer 345519 1906TCCACATGGAGTTGCTGTTACA PS/MOE 5-10-7 miR-194 gapmer 345520 1771ACAAGCTTTTTGCTCGTCTTAT PS/MOE 5-10-7 miR-208 gapmer 345521 938AGACACGTGCACTGTAGA PS/MOE 5-10-3 miR-139 gapmer 345522 1887GTCATCATTACCAGGCAGTATTA PS/MOE 5-10-8 miR-200b gapmer 345523 1831CATCGTTACCAGACAGTGTTA PS/MOE 5-10-6 miR-200a gapmer 345524 1827CAGCTTTCAAAATGATCTCAC PS/MOE 5-10-6 miR-Bantam gapmer 345922 1783ACAGTGCTTCATCTCA PO/6MOE-10deoxy mir-143 hemimer 345923 1848CTACAGTGCTTCATCTC PO/6MOE-11deoxy mir-143 hemimer 345924 1876GCTACAGTGCTTCATCT PO/6MOE-11deoxy mir-143 hemimer 345925 1875GCTACAGTGCTTCATC PO/6MOE-10deoxy mir-143 hemimer 345926 1803AGCTACAGTGCTTCAT PO/6MOE-10deoxy mir-143 hemimer 345927 1863GAGCTACAGTGCTTCA PO/6MOE-10deoxy mir-143 hemimer 345928 1916TGAGCTACAGTGCTTC PO/6MOE-10deoxy mir-143 hemimer 346685 1884GGCGGAACTTAGCCACTGTGAA PS/MOE miR-27a (RFAM- Human) 346686 1857CTTCAGTTATCACAGTACTGTA PS/MOE miR-101 (RFAM- Human) 346687 1802AGCAAGCCCAGACCGCAAAAAG PS/MOE miR-129b (RFAM- Human) 346688 1898TAGTTGGCAAGTCTAGAACCA PS/MOE miR-182* (RFAM- Human) 346689 1830CATCATTACCAGGCAGTATTAGAG PS/MOE miR-200a (RFAM- Human) 346690 1792ACTGATATCAGCTCAGTAGGCAC PS/MOE miR-189 (RFAM- Human) 346691 1870GCAGAAGCATTTCCACACAC PS/MOE miR-147 (RFAM- Human) 346692 1889TAAACGGAACCACTAGTGACTTG PS/MOE miR-224 (RFAM- Human) 346693 1838CCCTCTGGTCAACCAGTCACA PS/MOE miR-134 (RFAM- Human) 346694 1763AACCCATGGAATTCAGTTCTCA PS/MOE miR-146 (RFAM- Human) 346695 1824CACTGGTACAAGGGTTGGGAGA PS/MOE miR-150 (RFAM- Human) 346696 1893TACCTGCACTATAAGCACTTTA PS/MOE mir-20 346697 1788 ACCTATCCTGAATTACTTGAAPS/MOE mir-26b 346698 1793 ACTGATTTCAAATGGTGCTA PS/MOE mir-29b 3466991847 CGGCTGCAACACAAGACACGA PS/MOE miR-187 (RFAM- Human) 346700 1844CGACCATGGCTGTAGACTGTTA PS/MOE miR-132 (RFAM- Human) 346701 1901TCACATAGGAATAAAAAGCCATA PS/MOE miR-135 (RFAM- Human) 346702 1893TACCTGCACTATAAGCACTTTA PS/MOE 5-10-7 mir-20 gapmer 346703 1788ACCTATCCTGAATTACTTGAA PS/MOE 5-10-6 mir-26b gapmer 346704 1884GGCGGAACTTAGCCACTGTGAA PS/MOE 5-10-7 miR-27a (RFAM- gapmer Human) 3467051857 CTTCAGTTATCACAGTACTGTA PS/MOE 5-10-7 miR-101 (RFAM- gapmer Human)346706 1793 ACTGATTTCAAATGGTGCTA PS/MOE 5-10-5 mir-29b gapmer 3467071847 CGGCTGCAACACAAGACACGA PS/MOE 5-10-6 miR-187 (RFAM- gapmer Human)346708 1844 CGACCATGGCTGTAGACTGTTA PS/MOE 5-10-7 miR-132 (RFAM- gapmerHuman) 346709 1901 TCACATAGGAATAAAAAGCCATA PS/MOE 5-10-8 miR-135 (RFAM-gapmer Human) 346710 1802 AGCAAGCCCAGACCGCAAAAAG PS/MOE 5-10-7 miR-129b(RFAM- gapmer Human) 346711 1898 TAGTTGGCAAGTCTAGAACCA PS/MOE 5-10-6miR-182* (RFAM- gapmer Human) 346712 1830 CATCATTACCAGGCAGTATTAGAGPS/MOE 5-10-9 miR-200a (RFAM- gapmer Human) 346713 1792ACTGATATCAGCTCAGTAGGCAC PS/MOE 5-10-8 miR-189 (RFAM- gapmer Human)346714 1870 GCAGAAGCATTTCCACACAC PS/MOE 5-10-5 miR-147 (RFAM- gapmerHuman) 346715 1889 TAAACGGAACCACTAGTGACTTG PS/MOE 5-10-8 miR-224 (RFAM-gapmer Human) 346716 1838 CCCTCTGGTCAACCAGTCACA PS/MOE 5-10-6 miR-134(RFAM- gapmer Human) 346717 1763 AACCCATGGAATTCAGTTCTCA PS/MOE 5-10-7miR-146 (RFAM- gapmer Human) 346718 1824 CACTGGTACAAGGGTTGGGAGA PS/MOE5-10-7 miR-150 (RFAM- gapmer Human) 346905 1907 TCCAGTCAAGGATGTTTACAPS/MOE miR-30e (RFAM- M. musculus) 346906 1781 ACAGGATTGAGGGGGGGCCCTPS/MOE miR-296 (RFAM- M. musculus) 346907 1815 ATGTATGTGGGACGGTAAACCAPS/MOE miR-299 (RFAM- M. musculus) 346908 1881 GCTTTGACAATACTATTGCACTGPS/MOE miR-301 (RFAM- M. musculus) 346909 1902 TCACCAAAACATGGAAGCACTTAPS/MOE miR-302 (RFAM- M. musculus) 346910 1866 GCAATCAGCTAACTACACTGCCTPS/MOE miR-34a (RFAM- M. musculus) 346911 1776 ACACTGATTTCAAATGGTGCTAPS/MOE miR-29b (RFAM- M. musculus) 346912 1851 CTAGTGGTCCTAAACATTTCAPS/MOE miR-203 (RFAM- M. musculus) 346913 1795 AGAAAGGCAGCAGGTCGTATAGPS/MOE let-7d* (RFAM- M. musculus) 346914 1810 ATCTGCACTGTCAGCACTTTAPS/MOE miR-106b (RFAM- M. musculus) 346915 1784 ACATCGTTACCAGACAGTGTTAPS/MOE miR-200a (RFAM- M. musculus) 346916 1874 GCGGAACTTAGCCACTGTGAAPS/MOE miR-27a (RFAM- M. musculus) 346917 1826 CAGCTATGCCAGCATCTTGCCTPS/MOE miR-31 (RFAM-M. musculus) 346918 1829 CAGGCCGGGACAAGTGCAATAPS/MOE miR-92 (RFAM-M. musculus) 346919 1849 CTACCTGCACGAACAGCACTTTGPS/MOE miR-93 (RFAM-M. musculus) 346920 1801 AGCAAAAATGTGCTAGTGCCAAAPS/MOE miR-96 (RFAM-M. musculus) 346921 1759 AACAACCAGCTAAGACACTGCCAPS/MOE miR-172 (RFAM- M. musculus) 346922 1907 TCCAGTCAAGGATGTTTACAPS/MOE 5-10-5 miR-30e (RFAM- gapmer M. musculus) 346923 1781ACAGGATTGAGGGGGGGCCCT PS/MOE 5-10-6 miR-296 (RFAM- gapmer M. musculus)346924 1815 ATGTATGTGGGACGGTAAACCA PS/MOE 5-10-7 miR-299 (RFAM- gapmerM. musculus) 346925 1881 GCTTTGACAATACTATTGCACTG PS/MOE 5-10-8 miR-301(RFAM- gapmer M. musculus) 346926 1902 TCACCAAAACATGGAAGCACTTA PS/MOE5-10-8 miR-302 (RFAM- gapmer M. musculus) 346927 1866GCAATCAGCTAACTACACTGCCT PS/MOE 5-10-8 miR-34a (RFAM- gapmer M. musculus)346928 1776 ACACTGATTTCAAATGGTGCTA PS/MOE 5-10-7 miR-29b (RFAM- gapmerM. musculus) 346929 1851 CTAGTGGTCCTAAACATTTCA PS/MOE 5-10-6 miR-203(RFAM- gapmer M. musculus) 346930 1795 AGAAAGGCAGCAGGTCGTATAG PS/MOE5-10-7 let-7d* (RFAM- gapmer M. musculus) 346931 1810ATCTGCACTGTCAGCACTTTA PS/MOE 5-10-6 miR-106b (RFAM- gapmer M. musculus)346932 1784 ACATCGTTACCAGACAGTGTTA PS/MOE 5-10-7 miR-200a (RFAM- gapmerM. musculus) 346933 1874 GCGGAACTTAGCCACTGTGAA PS/MOE 5-10-6 miR-27a(RFAM- gapmer M. musculus) 346934 1826 CAGCTATGCCAGCATCTTGCCT PS/MOE5-10-7 miR-31 (RFAM-M. musculus) gapmer 346935 1829CAGGCCGGGACAAGTGCAATA PS/MOE 5-10-6 miR-92 (RFAM-M. musculus) gapmer346936 1849 CTACCTGCACGAACAGCACTTTG PS/MOE 5-10-8 miR-93 (RFAM-M.musculus) gapmer 346937 1801 AGCAAAAATGTGCTAGTGCCAAA PS/MOE 5-10-8miR-96 (RFAM-M. musculus) gapmer 346938 1759 AACAACCAGCTAAGACACTGCCAPS/MOE 5-10-8 miR-172 (RFAM- gapmer M. musculus) 347385 1782ACAGTGCTTCATCTC PO/6MOE-9deoxy mir-143 hemimer 347386 1848CTACAGTGCTTCATCTC PO/6MOE-11deoxy mir-143 hemimer 347387 1876GCTACAGTGCTTCATCT PO/6MOE-11deoxy mir-143 hemimer 347388 1875GCTACAGTGCTTCATC PO/6MOE-10deoxy mir-143 hemimer 347389 1803AGCTACAGTGCTTCAT PO/6MOE-10deoxy mir-143 hemimer 347390 1863GAGCTACAGTGCTTCA PO/6MOE-10deoxy mir-143 hemimer 347391 1916TGAGCTACAGTGCTTC PO/6MOE-10deoxy mir-143 hemimer 347452 1783ACAGTGCTTCATCTCA PO/6MOE-10deoxy mir-143 hemimer 347453 1783ACAGTGCTTCATCTCA PO/6MOE-10deoxy mir-143 hemimer 348116 1922TTCGCCCTCTCAACCCAGCTTTT PS/MOE miR-320 348117 1860 GAACCCACAATCCCTGGCTTAPS/MOE miR-321-1 348118 1886 GTAAACCATGATGTGCTGCTA PS/MOE miR-15b(Michael et al) 348119 1908 TCCATAAAGTAGGAAACACTACA PS/MOE miR-142as(Michael et al) 348120 1864 GAGCTACAGTGCTTCATCTCA PS/MOE miR-143(Michael et al) 348121 1883 GGATTCCTGGGAAAACTGGAC PS/MOE miR-145(Michael et al) 348122 1905 TCATCATTACCAGGCAGTATTA PS/MOE miR-200b(Michael et al) 348123 1791 ACTATACAATCTACTACCTCA PS/MOE let-7f (Michaelet al) 348124 1820 CACAAATTCGGTTCTACAGGGTA PS/MOE miR-10b (Michael etal) 348125 1878 GCTGGATGCAAACCTGCAAAACT PS/MOE miR-19b (Michael et al)348126 1873 GCCTATCCTGGATTACTTGAA PS/MOE miR-26a (Michael et al) 3481271869 GCAGAACTTAGCCACTGTGAA PS/MOE miR-27* (Michael et al) 348128 1858CTTCCAGTCAAGGATGTTTACA PS/MOE miR-97 (Michael et al) 348129 1855CTGGCTGTCAATTCATAGGTCA PS/MOE miR-192 (Michael et al) 348130 1922TTCGCCCTCTCAACCCAGCTTTT PS/MOE 5-10-8 miR-320 gapmer 348131 1860GAACCCACAATCCCTGGCTTA PS/MOE 5-10-6 miR-321-1 gapmer 348132 1886GTAAACCATGATGTGCTGCTA PS/MOE 5-10-6 miR-15b gapmer (Michael et al)348133 1908 TCCATAAAGTAGGAAACACTACA PS/MOE 5-10-8 miR-142as gapmer(Michael et al) 348134 1864 GAGCTACAGTGCTTCATCTCA PS/MOE 5-10-6 miR-143gapmer (Michael et al) 348135 1883 GGATTCCTGGGAAAACTGGAC PS/MOE 5-10-6miR-145 gapmer (Michael et al) 348136 1905 TCATCATTACCAGGCAGTATTA PS/MOE5-10-7 miR-200b gapmer (Michael et al) 348137 1791 ACTATACAATCTACTACCTCAPS/MOE 5-10-6 let-7f (Michael gapmer et al) 348138 1820CACAAATTCGGTTCTACAGGGTA PS/MOE 5-10-8 miR-10b gapmer (Michael et al)348139 1878 GCTGGATGCAAACCTGCAAAACT PS/MOE 5-10-8 miR-19b gapmer(Michael et al) 348140 1873 GCCTATCCTGGATTACTTGAA PS/MOE 5-10-6 miR-26agapmer (Michael et al) 348141 1869 GCAGAACTTAGCCACTGTGAA PS/MOE 5-10-6miR-27* gapmer (Michael et al) 348142 1858 CTTCCAGTCAAGGATGTTTACA PS/MOE5-10-7 miR-97 (Michael gapmer et al) 348143 1855 CTGGCTGTCAATTCATAGGTCAPS/MOE 5-10-7 miR-192 gapmer (Michael et al) 354040 1751AAACCACACAACCTACTACCTCA PS/MOE let-7b-Ruvkun 354041 1752AAACCATACAACCTACTACCTCA PS/MOE let-7c-Ruvkun 354042 1764AACTATGCAACCTACTACCTCT PS/MOE let-7d-Ruvkun 354043 1765AACTGTACAAACTACTACCTCA PS/MOE let-7gL-Ruvkun 354044 1760AACAGCACAAACTACTACCTCA PS/MOE let-7i-Ruvkun 354045 1924TTGGCATTCACCGCGTGCCTTAA PS/MOE mir-124a-Ruvkun 354046 1833CCAAGCTCAGACGGATCCGA PS/MOE mir-127-Ruvkun 354047 1896TACTTTCGGTTATCTAGCTTTA PS/MOE mir-131-Ruvkun 354048 1846CGGCCTGATTCACAACACCAGCT PS/MOE mir-138-Ruvkun 354049 1768ACAAACCATTATGTGCTGCTA PS/MOE mir-15-Ruvkun 354050 1789ACGCCAATATTTACGTGCTGCTA PS/MOE mir-16-Ruvkun 354051 1852CTATCTGCACTAGATGCACCTTA PS/MOE mir-18-Ruvkun 354052 1779ACAGCTGCTTTTGGGATTCCGTTG PS/MOE mir-191-Ruvkun 354053 1891TAACCGATTTCAGATGGTGCTA PS/MOE mir-29a-Ruvkun 354054 1813ATGCTTTGACAATACTATTGCACTG PS/MOE mir-301-Ruvkun 354055 1805AGCTGAGTGTAGGATGTTTACA PS/MOE mir-30b-Ruvkun 354056 1804AGCTGAGAGTGTAGGATGTTTACA PS/MOE mir-30c-Ruvkun 354057 1807AGCTTCCAGTCGGGGATGTTTACA PS/MOE mir-30d-Ruvkun 354058 1835CCAGCAGCACCTGGGGCAGTGG PS/MOE mir-324-3p- Ruvkun 354059 1899TATGGCAGACTGTGATTTGTTG PS/MOE mir-7-1*-Ruvkun 354060 1850CTACCTGCACTGTAAGCACTTTG PS/MOE mir-91-Ruvkun 354061 1822CACATAGGAATGAAAAGCCATA PS/MOE mir-135b (Ruvkun) 354062 1895TACTAGACTGTGAGCTCCTCGA PS/MOE mir-151* (Ruvkun) 354063 1885GGCTATAAAGTAACTGAGACGGA PS/MOE mir-340 (Ruvkun) 354064 1923TTCTAGGATAGGCCCAGGGGC PS/MOE mir-331 (Ruvkun) 354065 1892TACATACTTCTTTACATTCCA PS/MOE miR-1 (RFAM) 354066 1817CAATCAGCTAACTACACTGCCT PS/MOE miR-34c (RFAM) 354067 1837CCCCTATCACGATTAGCATTAA PS/MOE miR-155 (RFAM) 354068 1910TCCATCATTACCCGGCAGTATT PS/MOE miR-200c (RFAM) 354069 1818CAATCAGCTAATGACACTGCCT PS/MOE miR-34b (RFAM) 354070 1753AAACCCAGCAGACAATGTAGCT PS/MOE mir-221 (RFAM- M. musculus) 354071 1796AGACCCAGTAGCCAGATGTAGCT PS/MOE mir-222 (RFAM- M. musculus) 354072 1917TGAGCTCCTGGAGGACAGGGA PS/MOE mir-339-1 (RFAM) 354073 1925TTTAAGTGCTCATAATGCAGT PS/MOE miR-20* (human) 354074 1926TTTTCCCATGCCCTATACCTCT PS/MOE miR-202 (human) 354075 1856CTTCAGCTATCACAGTACTGTA PS/MOE miR-101b 354076 1894TACCTGCACTGTTAGCACTTTG PS/MOE miR-106a 354077 1772 ACAAGTGCCCTCACTGCAGTPS/MOE miR-17-3p 354078 1859 GAACAGGTAGTCTAAACACTGGG PS/MOE miR-199b(mouse) 354079 1915 TCTTCCCATGCGCTATACCTCT PS/MOE miR-202 (mouse) 3540801808 AGGCAAAGGATGACAAAGGGAA PS/MOE miR-211 (mouse) 354081 1809ATCCAGTCAGTTCCTGATGCAGTA PS/MOE miR-217 (mouse) 354082 1888TAAACGGAACCACTAGTGACTTA PS/MOE miR-224 (RFAM mouse) 354083 1758AACAAAATCACAAGTCTTCCA PS/MOE miR-7b 354084 1919 TGTAAGTGCTCGTAATGCAGTPS/MOE miR-20* (mouse) 354085 1778 ACACTTACTGGACACCTACTAGG PS/MOEmir-325 (human) 354086 1777 ACACTTACTGAGCACCTACTAGG PS/MOE mir-325(mouse) 354087 1877 GCTGGAGGAAGGGCCCAGAGG PS/MOE mir-326 (human) 3540881794 ACTGGAGGAAGGGCCCAGAGG PS/MOE mir-326 (mouse) 354089 1755AAAGAGGTTAACCAGGTGTGTT PS/MOE mir-329-1 (human) 354090 1750AAAAAGGTTAGCTGGGTGTGTT PS/MOE mir-329-1 (mouse) 354091 1914TCTCTGCAGGCCGTGTGCTTTGC PS/MOE mir-330 (human) 354092 1913TCTCTGCAGGCCCTGTGCTTTGC PS/MOE mir-330 (mouse) 354093 1757AAAGGCATCATATAGGAGCTGGA PS/MOE mir-337 (human) 354094 1756AAAGGCATCATATAGGAGCTGAA PS/MOE mir-337 (mouse) 354095 1872GCCCTGGACTAGGAGTCAGCA PS/MOE mir-345 (human) 354096 1868GCACTGGACTAGGGGTCAGCA PS/MOE mir-345 (mouse) 354097 1799AGAGGCAGGCATGCGGGCAGACA PS/MOE mir-346 (human) 354098 1798AGAGGCAGGCACTCGGGCAGACA PS/MOE mir-346 (mouse) 354099 1840CCTCAAGGAGCCTCAGTCTAG PS/MOE miR-151 (mouse) 354100 1841CCTCAAGGAGCCTCAGTCTAGT PS/MOE miR-151 (rat) 354101 1797AGAGGCAGGCACTCAGGCAGACA PS/MOE miR-346 (rat) 354102 1819CAATCAGCTAATTACACTGCCTA PS/MOE miR-34b (mouse) 354103 1842CCTCAAGGAGCTTCAGTCTAGT PS/MOE miR-151 (hum) 354104 1751AAACCACACAACCTACTACCTCA PS/MOE 5-10-8 let-7b-Ruvkun gapmer 354105 1752AAACCATACAACCTACTACCTCA PS/MOE 5-10-8 let-7c-Ruvkun gapmer 354106 1764AACTATGCAACCTACTACCTCT PS/MOE 5-10-7 let-7d-Ruvkun gapmer 354107 1765AACTGTACAAACTACTACCTCA PS/MOE 5-10-7 let-7gL-Ruvkun gapmer 354108 1760AACAGCACAAACTACTACCTCA PS/MOE 5-10-7 let-7i-Ruvkun gapmer 354109 1924TTGGCATTCACCGCGTGCCTTAA PS/MOE 5-10-8 mir-124a-Ruvkun gapmer 354110 1833CCAAGCTCAGACGGATCCGA PS/MOE 5-10-5 mir-127-Ruvkun gapmer 354111 1896TACTTTCGGTTATCTAGCTTTA PS/MOE 5-10-7 mir-131-Ruvkun gapmer 354112 1846CGGCCTGATTCACAACACCAGCT PS/MOE 5-10-8 mir-138-Ruvkun gapmer 354113 1768ACAAACCATTATGTGCTGCTA PS/MOE 5-10-6 mir-15-Ruvkun gapmer 354114 1789ACGCCAATATTTACGTGCTGCTA PS/MOE 5-10-8 mir-16-Ruvkun gapmer 354115 1852CTATCTGCACTAGATGCACCTTA PS/MOE 5-10-8 mir-18-Ruvkun gapmer 354116 1779ACAGCTGCTTTTGGGATTCCGTTG PS/MOE 5-10-9 mir-191-Ruvkun gapmer 354117 1891TAACCGATTTCAGATGGTGCTA PS/MOE 5-10-7 mir-29a-Ruvkun gapmer 354118 1813ATGCTTTGACAATACTATTGCACTG PS/MOE 5-10-10 mir-301-Ruvkun gapmer 3541191805 AGCTGAGTGTAGGATGTTTACA PS/MOE 5-10-7 mir-30b-Ruvkun gapmer 3541201804 AGCTGAGAGTGTAGGATGTTTACA PS/MOE 5-10-9 mir-30c-Ruvkun gapmer 3541211807 AGCTTCCAGTCGGGGATGTTTACA PS/MOE 5-10-9 mir-30d-Ruvkun gapmer 3541221835 CCAGCAGCACCTGGGGCAGTGG PS/MOE 5-10-7 mir-324-3p- gapmer Ruvkun354123 1899 TATGGCAGACTGTGATTTGTTG PS/MOE 5-10-7 mir-7-1*-Ruvkun gapmer354124 1850 CTACCTGCACTGTAAGCACTTTG PS/MOE 5-10-8 mir-91-Ruvkun gapmer354125 1822 CACATAGGAATGAAAAGCCATA PS/MOE 5-10-7 mir-135b gapmer(Ruvkun) 354126 1895 TACTAGACTGTGAGCTCCTCGA PS/MOE 5-10-7 mir-151*gapmer (Ruvkun) 354127 1885 GGCTATAAAGTAACTGAGACGGA PS/MOE 5-10-8mir-340 gapmer (Ruvkun) 354128 1923 TTCTAGGATAGGCCCAGGGGC PS/MOE 5-10-6mir-331 gapmer (Ruvkun) 354129 1892 TACATACTTCTTTACATTCCA PS/MOE 5-10-6miR-1 (RFAM) gapmer 354130 1817 CAATCAGCTAACTACACTGCCT PS/MOE 5-10-7miR-34c (RFAM) gapmer 354131 1837 CCCCTATCACGATTAGCATTAA PS/MOE 5-10-7miR-155 (RFAM) gapmer 354132 1910 TCCATCATTACCCGGCAGTATT PS/MOE 5-10-7miR-200c (RFAM) gapmer 354133 1818 CAATCAGCTAATGACACTGCCT PS/MOE 5-10-7miR-34b (RFAM) gapmer 354134 1753 AAACCCAGCAGACAATGTAGCT PS/MOE 5-10-7mir-221 (RFAM- gapmer M. musculus) 354135 1796 AGACCCAGTAGCCAGATGTAGCTPS/MOE 5-10-8 mir-222 (RFAM- gapmer M. musculus) 354136 1917TGAGCTCCTGGAGGACAGGGA PS/MOE 5-10-6 mir-339-1 gapmer (RFAM) 354137 1925TTTAAGTGCTCATAATGCAGT PS/MOE 5-10-6 miR-20* (human) gapmer 354138 1926TTTTCCCATGCCCTATACCTCT PS/MOE 5-10-7 miR-202 (human) gapmer 354139 1856CTTCAGCTATCACAGTACTGTA PS/MOE 5-10-7 miR-101b gapmer 354140 1894TACCTGCACTGTTAGCACTTTG PS/MOE 5-10-7 miR-106a gapmer 354141 1772ACAAGTGCCCTCACTGCAGT PS/MOE 5-10-5 miR-17-3p gapmer 354142 1859GAACAGGTAGTCTAAACACTGGG PS/MOE 5-10-8 miR-199b gapmer (mouse) 3541431915 TCTTCCCATGCGCTATACCTCT PS/MOE 5-10-7 miR-202 (mouse) gapmer 3541441808 AGGCAAAGGATGACAAAGGGAA PS/MOE 5-10-7 miR-211 (mouse) gapmer 3541451809 ATCCAGTCAGTTCCTGATGCAGTA PS/MOE 5-10-9 miR-217 (mouse) gapmer354146 1888 TAAACGGAACCACTAGTGACTTA PS/MOE 5-10-8 miR-224 (RFAM gapmermouse) 354147 1758 AACAAAATCACAAGTCTTCCA PS/MOE 5-10-6 miR-7b gapmer354148 1919 TGTAAGTGCTCGTAATGCAGT PS/MOE 5-10-6 miR-20* (mouse) gapmer354149 1778 ACACTTACTGGACACCTACTAGG PS/MOE 5-10-8 mir-325 (human) gapmer354150 1777 ACACTTACTGAGCACCTACTAGG PS/MOE 5-10-8 mir-325 (mouse) gapmer354151 1877 GCTGGAGGAAGGGCCCAGAGG PS/MOE 5-10-6 mir-326 (human) gapmer354152 1794 ACTGGAGGAAGGGCCCAGAGG PS/MOE 5-10-6 mir-326 (mouse) gapmer354153 1755 AAAGAGGTTAACCAGGTGTGTT PS/MOE 5-10-7 mir-329-1 gapmer(human) 354154 1750 AAAAAGGTTAGCTGGGTGTGTT PS/MOE 5-10-7 mir-329-1gapmer (mouse) 354155 1914 TCTCTGCAGGCCGTGTGCTTTGC PS/MOE 5-10-8 mir-330(human) gapmer 354156 1913 TCTCTGCAGGCCCTGTGCTTTGC PS/MOE 5-10-8 mir-330(mouse) gapmer 354157 1757 AAAGGCATCATATAGGAGCTGGA PS/MOE 5-10-8 mir-337(human) gapmer 354158 1756 AAAGGCATCATATAGGAGCTGAA PS/MOE 5-10-8 mir-337(mouse) gapmer 354159 1872 GCCCTGGACTAGGAGTCAGCA PS/MOE 5-10-6 mir-345(human) gapmer 354160 1868 GCACTGGACTAGGGGTCAGCA PS/MOE 5-10-6 mir-345(mouse) gapmer 354161 1799 AGAGGCAGGCATGCGGGCAGACA PS/MOE 5-10-8 mir-346(human) gapmer 354162 1798 AGAGGCAGGCACTCGGGCAGACA PS/MOE 5-10-8 mir-346(mouse) gapmer 354163 1840 CCTCAAGGAGCCTCAGTCTAG PS/MOE 5-10-6 miR-151(mouse) gapmer 354164 1841 CCTCAAGGAGCCTCAGTCTAGT PS/MOE 5-10-7 miR-151(rat) gapmer 354165 1797 AGAGGCAGGCACTCAGGCAGACA PS/MOE 5-10-8 miR-346(rat) gapmer 354166 1819 CAATCAGCTAATTACACTGCCTA PS/MOE 5-10-8 miR-34b(mouse) gapmer 354167 1842 CCTCAAGGAGCTTCAGTCTAGT PS/MOE 5-10-7 miR-151(human) gapmer

In accordance with the present invention, oligomeric compounds weredesigned to mimic one or more miRNAs, pre-miRNAs or pri-miRNAs. Theoligomeric compounds of the present invention can also be designed tomimic a pri-miRNA, a pre-miRNA or a single- or double-stranded miRNAwhile incorporating certain chemical modifications that alter one ormore properties of the mimic, thus creating a construct with superiorproperties over the endogenous pri-miRNA, pre-miRNA or miRNA. Oligomericcompounds representing synthesized miRNAs or chemically modified miRNAmimics were given internal numerical identifiers (ISIS Numbers) and areshown in Table 66. These oligomeric compounds can be analyzed for theireffect on miRNA, pre-miRNA or pri-miRNA levels or for their effect ondownstream target RNA transcripts by quantitative real-time PCR or theycan be used in other assays to investigate the role of miRNAs or miRNAdownstream targets. In Table 66, “pri-miRNA” indicates the particularpri-miRNA from which the mature miRNA is normally processed when itoccurs in the cellular environment. All compounds listed in Table 66 areribonucleotides. The miRNA mimics consist of phosphorothioateinternucleoside linkages, indicated by “PS” in the “Chemistry” column ofTable 66, whereas synthesized miRNA oligomeric compounds withphosphodiester internucleoside linkages are indicated by “PO.”

TABLE 66 miRNAs and miRNA mimics SEQ ID Linkage ISIS # NO sequencechemistry Pri-miRNA 343092 437 ACCGAACAAAGTCTGACAGGA PO hypotheticalmiRNA-180 343098 1780 ACAGGAGTCTGAGCATTTGA PO miR-105 (Mourelatos)343099 1882 GGAACTTAGCCACTGTGAA PO miR-27 (Mourelatos) 343101 855TGCTCAATAAATACCCGTTGAA PO miR-95 (Mourelatos) 343102 1821CACAAGATCGGATCTACGGGTT PO miR-99 (Mourelatos) 343103 1903TCAGACCGAGACAAGTGCAATG PO miR-25 (Tuschl) 343104 1853CTCAATAGACTGTGAGCTCCTT PO miR-28 (Tuschl) 343105 1825CAGCTATGCCAGCATCTTGCC PO miR-31 (Tuschl) 343106 1865GCAACTTAGTAATGTGCAATA PO miR-32 (Tuschl) 343107 854GGAGTGAAGACACGGAGCCAGA PO miR-149 343108 1845 CGCAAGGTCGGTTCTACGGGTG POmiR-99b 343109 852 CACAGGTTAAAGGGTCTCAGGGA PO miR-125a 343110 853AGCCAAGCTCAGACGGATCCGA PO miR-127 343111 1909 TCCATCATCAAAACAAATGGAGT POmiR-136 343112 1843 CGAAGGCAACACGGATAACCTA PO miR-154 343113 1880GCTTCCAGTCGAGGATGTTTACA PO miR-30a-s 343114 1911 TCCGTGGTTCTACCCTGTGGTAPO miR-140-as 343115 1836 CCATAAAGTAGGAAACACTACA PO miR-142-as 3431171762 AACCAATGTGCAGACTACTGTA PO miR-199-as 343118 1904TCATACAGCTAGATAACCAAAGA PO miR-9 343119 1773 ACAAGTGCCTTCACTGCAGT POmiR-17 343120 1871 GCATTATTACTCACGGTACGA PO miR-126a 343121 1787ACCTAATATATCAAACATATCA PO miR-190 343122 1766 AAGCCCAAAAGGAGAATTCTTTG POmiR-186 343123 1839 CCTATCTCCCCTCTGGACC PO miR-198a 343124 1806AGCTGCTTTTGGGATTCCGTTG PO miR-191c 343125 760 CCACACACTTCCTTACATTCCA POmiR-206d 343126 761 ATCTGCACTGTCAGCACTTT PO miR-94 343127 762ACCCTTATCAGTTCTCCGTCCA PO miR-184 343128 763 GCCAATATTTCTGTGCTGCTA POmiR-195 343129 764 CTGGGACTTTGTAGGCCAGTT PO miR-193 343130 1861GAACTGCCTTTCTCTCCA PO miR-185 343131 1786 ACCCTCCACCATGCAAGGGATG POmiR-188 343132 1879 GCTGGGTGGAGAAGGTGGTGAA PO miR-197a 343133 1906TCCACATGGAGTTGCTGTTACA PO miR-194 343134 1771 ACAAGCTTTTTGCTCGTCTTAT POmiR-208 343135 938 AGACACGTGCACTGTAGA PO miR-139 343136 1887GTCATCATTACCAGGCAGTATTA PO miR-200b 343137 1831 CATCGTTACCAGACAGTGTTA POmiR-200a 343138 291 CTACCATAGGGTAAAACCACT PS mir-140 343139 292GCTGCAAACATCCGACTGAAAG PS mir-30a 343140 293 ACAACCAGCTAAGACACTGCCA PSmir-34 343141 294 AACACTGATTTCAAATGGTGCTA PS mir-29b 343142 295CGCCAATATTTACGTGCTGCTA PS mir-16 343143 296 CTAGTGGTCCTAAACATTTCAC PSmir-203 343144 297 AACAAAATCACTAGTCTTCCA PS mir-7 343145 298ACAAATTCGGTTCTACAGGGTA PS mir-10b 343146 299 AAAAGAGACCGGTTCACTGTGA PSmir-128a 343147 300 TCACTTTTGTGACTATGCAA PS mir-153 343148 301CAGAACTTAGCCACTGTGAA PS mir-27b 343149 302 GCAAAAATGTGCTAGTGCCAAA PSmir-96 343150 303 ACTACCTGCACTGTAAGCACTTTG PS mir-17as/mir-91 343151 304CGCGTACCAAAAGTAATAATG PS mir-123/mir-126as 343152 305GCGACCATGGCTGTAGACTGTTA PS mir-132 343153 306 AATGCCCCTAAAAATCCTTAT PSmir-108 343154 307 GTGGTAATCCCTGGCAATGTGAT PS mir-23b 343155 308AGCACAAACTACTACCTCA PS let-7i 343156 309 GGCCGTGACTGGAGACTGTTA PSmir-212 343157 310 ACTTTCGGTTATCTAGCTTTA PS mir-131 343158 311AACCACACAACCTACTACCTCA PS let-7b 343159 312 ATACATACTTCTTTACATTCCA PSmir-1d 343160 313 ACAAACACCATTGTCACACTCCA PS mir-122a 343161 314ACAGTTCTTCAACTGGCAGCTT PS mir-22 343162 315 ACAGGCCGGGACAAGTGCAATA PSmir-92 343163 316 GTAGTGCTTTCTACTTTATG PS mir-142 343164 317CAGTGAATTCTACCAGTGCCATA PS mir-183 343165 318 CTGCCTGTCTGTGCCTGCTGT PSmir-214 343166 319 TGAGCTACAGTGCTTCATCTCA PS mir-143 343167 320GGCTGTCAATTCATAGGTCAG PS mir-192 343168 321 AACTATACAACCTACTACCTCA PSlet-7a 343169 322 ACTCACCGACAGCGTTGAATGTT PS mir-181a 343170 323CAGACTCCGGTGGAATGAAGGA PS mir-205 343171 324 TCATAGCCCTGTACAATGCTGCT PSmir-103 343172 325 AGCCTATCCTGGATTACTTGAA PS mir-26a 343173 326CAATGCAACTACAATGCAC PS mir-33a 343174 327 CCCAACAACATGAAACTACCTA PSmir-196 343175 328 TGATAGCCCTGTACAATGCTGCT PS mir-107 343176 329GCTACCTGCACTGTAAGCACTTTT PS mir-106 343177 330 AACTATACAATCTACTACCTCA PSlet-7f 343178 331 AACCGATTTCAAATGGTGCTAG PS mir-29c 343179 332GCCCTTTTAACATTGCACTG PS mir-130a 343180 333 ACATGGTTAGATCAAGCACAA PSmir-218 343181 334 TGGCATTCACCGCGTGCCTTAA PS mir-124a 343182 335TCAACATCAGTCTGATAAGCTA PS mir-21 343183 336 CTAGTACATCATCTATACTGTA PSmir-144 343184 337 GAAACCCAGCAGACAATGTAGCT PS mir-221 343185 338GAGACCCAGTAGCCAGATGTAGCT PS mir-222 343186 339 CTTCCAGTCGGGGATGTTTACA PSmir-30d 343187 340 TCAGTTTTGCATGGATTTGCACA PS mir-19b 343188 341GAAAGAGACCGGTTCACTGTGA PS mir-128b 343189 342 GCAAGCCCAGACCGCAAAAAG PSmir-129 343190 343 TAGCTGGTTGAAGGGGACCAA PS mir-133b 343191 344ACTATGCAACCTACTACCTCT PS let-7d 343192 345 TGTAAACCATGATGTGCTGCTA PSmir-15b 343193 346 AACCGATTTCAGATGGTGCTAG PS mir-29a 343194 347GAACAGATAGTCTAAACACTGGG PS mir-199b 343195 348 ACTATACAACCTCCTACCTCA PSlet-7e 343196 349 AACCATACAACCTACTACCTCA PS let-7c 343197 350AGGCATAGGATGACAAAGGGAA PS mir-204 343198 351 AAGGGATTCCTGGGAAAACTGGAC PSmir-145 343199 352 GGTACAATCAACGGTCGATGGT PS mir-213 343200 353CTACCTGCACTATAAGCACTTTA PS mir-20 343201 354 ACAGCTGGTTGAAGGGGACCAA PSmir-133a 343202 355 GATTCACAACACCAGCT PS mir-138 343203 356AACAATACAACTTACTACCTCA PS mir-98 343204 357 TCACAAGTTAGGGTCTCAGGGA PSmir-125b 343205 358 GAACAGGTAGTCTGAACACTGGG PS mir-199a 343206 359AACCCACCGACAGCAATGAATGTT PS mir-181b 343207 360 CCATCTTTACCAGACAGTGTT PSmir-141 343208 361 TATCTGCACTAGATGCACCTTA PS mir-18 343209 362AAAGTGTCAGATACGGTGTGG PS mir-220 343210 363 CTGTTCCTGCTGAACTGAGCCA PSmir-24 343211 364 AGGCGAAGGATGACAAAGGGAA PS mir-211 343212 365TCAGTTATCACAGTACTGTA PS mir-101 343213 366 GCTGAGTGTAGGATGTTTACA PSmir-30b 343214 367 CACAAATTCGGATCTACAGGGTA PS mir-10a 343215 368TCAGTTTTGCATAGATTTGCACA PS mir-19a 343216 369 CACAAACCATTATGTGCTGCTA PSmir-15a 343217 370 CTACGCGTATTCTTAAGCAATA PS mir-137 343218 371AGAATTGCGTTTGGACAATCA PS mir-219 343219 372 ACAAAGTTCTGTGATGCACTGA PSmir-148b 343220 373 GCCCTTTCATCATTGCACTG PS mir-130b 343221 374CACAGTTGCCAGCTGAGATTA PS mir-216 343222 375 CACAAGTTCGGATCTACGGGTT PSmir-100 343223 376 CCGGCTGCAACACAAGACACGA PS mir-187 343224 377CAGCCGCTGTCACACGCACAG PS mir-210 343225 378 GTCTGTCAATTCATAGGTCAT PSmir-215 343226 379 GGGGTATTTGACAAACTGACA PS mir-223 343227 380GCTGAGAGTGTAGGATGTTTACA PS mir-30c 343228 381 AACCTATCCTGAATTACTTGAA PSmir-26b 343229 382 CCAAGTTCTGTCATGCACTGA PS mir-152 343230 383ATCACATAGGAATAAAAAGCCATA PS mir-135 343231 384 ATCCAATCAGTTCCTGATGCAGTAPS mir-217 343232 385 ACTGTACAAACTACTACCTCA PS let-7g 343233 386CAATGCAACAGCAATGCAC PS mir-33b 343234 387 TGTGAGTTCTACCATTGCCAAA PSmir-182 343235 388 ACAAAGTTCTGTAGTGCACTGA PS mir-148a 343236 389GGAAATCCCTGGCAATGTGAT PS mir-23a 343237 390 ACTCACCGACAGGTTGAATGTT PSmir-181c 343238 391 ACTGTAGGAATATGTTTGATA PS hypothetical miRNA-013343239 392 ATTAAAAAGTCCTCTTGCCCA PS hypothetical miRNA-023 343240 393GCTGCCGTATATGTGATGTCA PS hypothetical miRNA-030 343241 394GGTAGGTGGAATACTATAACA PS hypothetical miRNA-033 343242 395TAAACATCACTGCAAGTCTTA PS hypothetical miRNA-039 343243 396TTGTAAGCAGTTTTGTTGACA PS hypothetical miRNA-040 343244 397TCACAGAGAAAACAACTGGTA PS hypothetical miRNA-041 343245 398CCTCTCAAAGATTTCCTGTCA PS hypothetical miRNA-043 343246 399TGTCAGATAAACAGAGTGGAA PS hypothetical miRNA-044 343247 400GAGAATCAATAGGGCATGCAA PS hypothetical miRNA-055 343248 401AAGAACATTAAGCATCTGACA PS hypothetical miRNA-058 343249 402AATCTCTGCAGGCAAATGTGA PS hypothetical miRNA-070 343250 403AAACCCCTATCACGATTAGCA PS hypothetical miRNA-071 343251 404GCCCCATTAATATTTTAACCA PS hypothetical miRNA-075 343252 405CCCAATATCAAACATATCA PS hypothetical miRNA-079 343253 406TATGATAGCTTCCCCATGTAA PS hypothetical miRNA-083 343254 407CCTCAATTATTGGAAATCACA PS hypothetical miRNA-088 343255 408ATTGATGCGCCATTTGGCCTA PS hypothetical miRNA-090 343256 409CTGTGACTTCTCTATCTGCCT PS hypothetical miRNA-099 343257 410AAACTTGTTAATTGACTGTCA PS hypothetical miRNA-101 343258 411AAAGAAGTATATGCATAGGAA PS hypothetical miRNA-105 343259 412GATAAAGCCAATAAACTGTCA PS hypothetical miRNA-107 343260 413TCCGAGTCGGAGGAGGAGGAA PS hypothetical miRNA-111 343261 414ATCATTACTGGATTGCTGTAA PS hypothetical miRNA-120 343262 415CAAAAATTATCAGCCAGTTTA PS hypothetical miRNA-137 343263 416AATCTCATTTTCATACTTGCA PS hypothetical miRNA-138 343264 417AGAAGGTGGGGAGCAGCGTCA PS hypothetical miRNA-142 343265 418CAAAATTGCAAGCAAATTGCA PS hypothetical miRNA-143 343266 419TCCACAAAGCTGAACATGTCT PS hypothetical miRNA-144 343267 420TATTATCAGCATCTGCTTGCA PS hypothetical miRNA-153 343268 421AATAACACACATCCACTTTAA PS hypothetical miRNA-154 343269 422AAGAAGGAAGGAGGGAAAGCA PS hypothetical miRNA-156 343270 423ATGACTACAAGTTTATGGCCA PS hypothetical miRNA-161 343271 424CAAAACATAAAAATCCTTGCA PS hypothetical miRNA-164 343272 425TTACAGGTGCTGCAACTGGAA PS hypothetical miRNA-166 343273 426AGCAGGTGAAGGCACCTGGCT PS hypothetical miRNA-168 343274 427TATGAAATGCCAGAGCTGCCA PS hypothetical miRNA-169 343275 428CCAAGTGTTAGAGCAAGATCA PS hypothetical miRNA-170 343276 429AACGATAAAACATACTTGTCA PS hypothetical miRNA-171 343277 430AGTAACTTCTTGCAGTTGGA PS hypothetical miRNA-172 343278 431AGCCTCCTTCTTCTCGTACTA PS hypothetical miRNA-173 343279 432ACCTCAGGTGGTTGAAGGAGA PS hypothetical miRNA-175 343280 433ATATGTCATATCAAACTCCTA PS hypothetical miRNA-176 343281 434GTGAGAGTAGCATGTTTGTCT PS hypothetical miRNA-177 343282 435TGAAGGTTCGGAGATAGGCTA PS hypothetical miRNA-178 343283 436AATTGGACAAAGTGCCTTTCA PS hypothetical miRNA-179 343284 437ACCGAACAAAGTCTGACAGGA PS hypothetical miRNA-180 343285 438AACTACTTCCAGAGCAGGTGA PS hypothetical miRNA-181 343286 439GTAAGCGCAGCTCCACAGGCT PS hypothetical miRNA-183 343287 440GAGCTGCTCAGCTGGCCATCA PS hypothetical miRNA-185 343288 441TACTTTTCATTCCCCTCACCA PS hypothetical miRNA-188 343289 236TAGCTTATCAGACTGATGTTGA PS miR-104 (Mourelatos) 343290 1780ACAGGAGTCTGAGCATTTGA PS miR-105 (Mourelatos) 343291 1882GGAACTTAGCCACTGTGAA PS miR-27 (Mourelatos) 343292 848CTACCTGCACGAACAGCACTTT PS miR-93 (Mourelatos) 343293 855TGCTCAATAAATACCCGTTGAA PS miR-95 (Mourelatos) 343294 1821CACAAGATCGGATCTACGGGTT PS miR-99 (Mourelatos) 343295 1903TCAGACCGAGACAAGTGCAATG PS miR-25 (Tuschl) 343296 1853CTCAATAGACTGTGAGCTCCTT PS miR-28 (Tuschl) 343297 1825CAGCTATGCCAGCATCTTGCC PS miR-31 (Tuschl) 343298 1865GCAACTTAGTAATGTGCAATA PS miR-32 (Tuschl) 343299 854GGAGTGAAGACACGGAGCCAGA PS miR-149 343300 1845 CGCAAGGTCGGTTCTACGGGTG PSmiR-99b 343301 852 CACAGGTTAAAGGGTCTCAGGGA PS miR-125a 343302 853AGCCAAGCTCAGACGGATCCGA PS miR-127 343303 1909 TCCATCATCAAAACAAATGGAGT PSmiR-136 343304 1843 CGAAGGCAACACGGATAACCTA PS miR-154 343305 1880GCTTCCAGTCGAGGATGTTTACA PS miR-30a-s 343306 1911 TCCGTGGTTCTACCCTGTGGTAPS miR-140-as 343307 1836 CCATAAAGTAGGAAACACTACA PS miR-142-as 3433081761 AACAGGTAGTCTGAACACTGGG PS miR-199-s 343309 1762AACCAATGTGCAGACTACTGTA PS miR-199-as 343310 1904 TCATACAGCTAGATAACCAAAGAPS miR-9 343311 1773 ACAAGTGCCTTCACTGCAGT PS miR-17 343312 1871GCATTATTACTCACGGTACGA PS miR-126a 343313 1787 ACCTAATATATCAAACATATCA PSmiR-190 343314 1766 AAGCCCAAAAGGAGAATTCTTTG PS miR-186 343315 1839CCTATCTCCCCTCTGGACC PS miR-198a 343316 1806 AGCTGCTTTTGGGATTCCGTTG PSmiR-191c 343317 760 CCACACACTTCCTTACATTCCA PS miR-206d 343318 761ATCTGCACTGTCAGCACTTT PS miR-94 343319 762 ACCCTTATCAGTTCTCCGTCCA PSmiR-184 343320 763 GCCAATATTTCTGTGCTGCTA PS miR-195 343321 764CTGGGACTTTGTAGGCCAGTT PS miR-193 343322 1861 GAACTGCCTTTCTCTCCA PSmiR-185 343323 1786 ACCCTCCACCATGCAAGGGATG PS miR-188 343324 1879GCTGGGTGGAGAAGGTGGTGAA PS miR-197a 343325 1906 TCCACATGGAGTTGCTGTTACA PSmiR-194 343326 1771 ACAAGCTTTTTGCTCGTCTTAT PS miR-208 343327 938AGACACGTGCACTGTAGA PS miR-139 343328 1887 GTCATCATTACCAGGCAGTATTA PSmiR-200b 343329 1831 CATCGTTACCAGACAGTGTTA PS miR-200a 344290 1774ACACAAATTCGGTTCTACAGGG PO miR-10b (Tuschl) 344292 1867 GCACGAACAGCACTTTGPO miR-93 (Tuschl) 344293 1770 ACAAGATCGGATCTACGGGT PO miR-99a (Tuschl)344297 1912 TCTAGTGGTCCTAAACATTTCA PO miR-203 (Tuschl) 344298 1828CAGGCATAGGATGACAAAGGGAA PO miR-204 (Tuschl) 344299 1767AATACATACTTCTTTACATTCCA PO miR-1d (Tuschl) 344300 1769ACAAATTCGGATCTACAGGGTA PS miR-10 (Tuschl) 344301 1774ACACAAATTCGGTTCTACAGGG PS miR-10b (Tuschl) 344302 1890TAACCGATTTCAAATGGTGCTA PS miR-29c (Tuschl) 344303 1867 GCACGAACAGCACTTTGPS miR-93 (Tuschl) 344304 1770 ACAAGATCGGATCTACGGGT PS miR-99a (Tuschl)344305 1816 CAAACACCATTGTCACACTCCA PS miR-122a, b (Tuschl) 344306 1920TGTCAATTCATAGGTCAG PS miR-192 (Tuschl) 344307 1832 CCAACAACATGAAACTACCTAPS miR-196 (Tuschl) 344308 1912 TCTAGTGGTCCTAAACATTTCA PS miR-203(Tuschl) 344309 1828 CAGGCATAGGATGACAAAGGGAA PS miR-204 (Tuschl) 3443101767 AATACATACTTCTTTACATTCCA PS miR-1d (Tuschl) 344354 1812ATGCCCTTTTAACATTGCACTG PO mir-130 (Kosik) 344356 1921TGTCCGTGGTTCTACCCTGTGGTA PO mir-239* (Kosik) 344358 1814ATGCTTTTTGGGGTAAGGGCTT PO mir-129as/mir-258* (Kosik) 344359 1811ATGCCCTTTCATCATTGCACTG PO mir-266* (Kosik) 344360 1918TGGCATTCACCGCGTGCCTTA PS mir-124a (Kosik) 344361 1754AAAGAGACCGGTTCACTGTGA PS mir-128 (Kosik) 344362 1812ATGCCCTTTTAACATTGCACTG PS mir-130 (Kosik) 344363 1854CTCACCGACAGCGTTGAATGTT PS mir-178 (Kosik) 344364 1921TGTCCGTGGTTCTACCCTGTGGTA PS mir-239* (Kosik) 344365 1823CACATGGTTAGATCAAGCACAA PS mir-253* (Kosik) 344366 1814ATGCTTTTTGGGGTAAGGGCTT PS mir-129as/mir-258* (Kosik) 344367 1811ATGCCCTTTCATCATTGCACTG PS mir-266* (Kosik) 344625 1785ACATTTTTCGTTATTGCTCTTGA PO mir-240* (Kosik) 344626 1790ACGGAAGGGCAGAGAGGGCCAG PO mir-232* (Kosik) 344627 1775ACACCAATGCCCTAGGGGATGCG PO mir-227* (Kosik) 344628 1834CCAGCAGCACCTGGGGCAGT PO mir-226* (Kosik) 344629 1900TCAACAAAATCACTGATGCTGGA PO mir-244* (Kosik) 344630 1800AGAGGTCGACCGTGTAATGTGC PO mir-224* (Kosik) 344631 1862GACGGGTGCGATTTCTGTGTGAGA PO mir-248* (Kosik) 344632 1785ACATTTTTCGTTATTGCTCTTGA PS mir-240* (Kosik) 344633 1790ACGGAAGGGCAGAGAGGGCCAG PS mir-232* (Kosik) 344634 1775ACACCAATGCCCTAGGGGATGCG PS mir-227* (Kosik) 344635 1834CCAGCAGCACCTGGGGCAGT PS mir-226* (Kosik) 344636 1900TCAACAAAATCACTGATGCTGGA PS mir-244* (Kosik) 344637 1800AGAGGTCGACCGTGTAATGTGC PS mir-224* (Kosik) 344638 1862GACGGGTGCGATTTCTGTGTGAGA PS mir-248* (Kosik) 345527 1827CAGCTTTCAAAATGATCTCAC PO miR-Bantam 345529 1897 TAGGAGAGAGAAAAAGACTGA PSmiR-14 345531 1827 CAGCTTTCAAAATGATCTCAC PS miR-Bantam 345708 1897TAGGAGAGAGAAAAAGACTGA PO miR-14 346721 1884 GGCGGAACTTAGCCACTGTGAA POmiR-27a (RFAM-Human) 346722 1857 CTTCAGTTATCACAGTACTGTA PO miR-101(RFAM-Human) 346727 1802 AGCAAGCCCAGACCGCAAAAAG PO miR-129b (RFAM-Human)346728 1898 TAGTTGGCAAGTCTAGAACCA PO miR-182* (RFAM-Human) 346729 1830CATCATTACCAGGCAGTATTAGAG PO miR-200a (RFAM-Human) 346730 1792ACTGATATCAGCTCAGTAGGCAC PO miR-189 (RFAM-Human) 346731 1870GCAGAAGCATTTCCACACAC PO miR-147 (RFAM-Human) 346732 1889TAAACGGAACCACTAGTGACTTG PO miR-224 (RFAM-Human) 346733 1838CCCTCTGGTCAACCAGTCACA PO miR-134 (RFAM-Human) 346734 1763AACCCATGGAATTCAGTTCTCA PO miR-146 (RFAM-Human) 346735 1824CACTGGTACAAGGGTTGGGAGA PO miR-150 (RFAM-Human) 346736 1893TACCTGCACTATAAGCACTTTA PS mir-20 346737 1788 ACCTATCCTGAATTACTTGAA PSmir-26b 346738 1884 GGCGGAACTTAGCCACTGTGAA PS miR-27a (RFAM-Human)346739 1857 CTTCAGTTATCACAGTACTGTA PS miR-101 (RFAM-Human) 346740 1793ACTGATTTCAAATGGTGCTA PS mir-29b 346741 1847 CGGCTGCAACACAAGACACGA PSmiR-187 (RFAM-Human) 346742 1844 CGACCATGGCTGTAGACTGTTA PS miR-132(RFAM-Human) 346743 1901 TCACATAGGAATAAAAAGCCATA PS miR-135 (RFAM-Human)346744 1802 AGCAAGCCCAGACCGCAAAAAG PS miR-129b (RFAM-Human) 346745 1898TAGTTGGCAAGTCTAGAACCA PS miR-182* (RFAM-Human) 346746 1830CATCATTACCAGGCAGTATTAGAG PS miR-200a (RFAM-Human) 346747 1792ACTGATATCAGCTCAGTAGGCAC PS miR-189 (RFAM-Human) 346748 1870GCAGAAGCATTTCCACACAC PS miR-147 (RFAM-Human) 346749 1889TAAACGGAACCACTAGTGACTTG PS miR-224 (RFAM-Human) 346750 1838CCCTCTGGTCAACCAGTCACA PS miR-134 (RFAM-Human) 346751 1763AACCCATGGAATTCAGTTCTCA PS miR-146 (RFAM-Human) 346752 1824CACTGGTACAAGGGTTGGGAGA PS miR-150 (RFAM-Human) 346939 1907TCCAGTCAAGGATGTTTACA PO miR-30e (RFAM-M. musculus) 346940 1781ACAGGATTGAGGGGGGGCCCT PO miR-296 (RFAM-M. musculus) 346941 1815ATGTATGTGGGACGGTAAACCA PO miR-299 (RFAM-M. musculus) 346942 1881GCTTTGACAATACTATTGCACTG PO miR-301 (RFAM-M. musculus) 346943 1902TCACCAAAACATGGAAGCACTTA PO miR-302 (RFAM-M. musculus) 346944 1866GCAATCAGCTAACTACACTGCCT PO miR-34a (RFAM-M. musculus) 346945 1776ACACTGATTTCAAATGGTGCTA PO miR-29b (RFAM-M. musculus) 346947 1795AGAAAGGCAGCAGGTCGTATAG PO let-7d* (RFAM-M. musculus) 346948 1810ATCTGCACTGTCAGCACTTTA PO miR-106b (RFAM-M. musculus) 346949 1784ACATCGTTACCAGACAGTGTTA PO miR-200a (RFAM-M. musculus) 346950 1874GCGGAACTTAGCCACTGTGAA PO miR-27a (RFAM-M. musculus) 346951 1826CAGCTATGCCAGCATCTTGCCT PO miR-31 (RFAM-M. musculus) 346954 1801AGCAAAAATGTGCTAGTGCCAAA PO miR-96 (RFAM-M. musculus) 346955 1759AACAACCAGCTAAGACACTGCCA PO miR-172 (RFAM-M. musculus) 346956 1907TCCAGTCAAGGATGTTTACA PS miR-30e (RFAM-M. musculus) 346957 1781ACAGGATTGAGGGGGGGCCCT PS miR-296 (RFAM-M. musculus) 346958 1815ATGTATGTGGGACGGTAAACCA PS miR-299 (RFAM-M. musculus) 346959 1881GCTTTGACAATACTATTGCACTG PS miR-301 (RFAM-M. musculus) 346960 1902TCACCAAAACATGGAAGCACTTA PS miR-302 (RFAM-M. musculus) 346961 1866GCAATCAGCTAACTACACTGCCT PS miR-34a (RFAM-M. musculus) 346962 1776ACACTGATTTCAAATGGTGCTA PS miR-29b (RFAM-M. musculus) 346963 1851CTAGTGGTCCTAAACATTTCA PS miR-203 (RFAM-M. musculus) 346964 1795AGAAAGGCAGCAGGTCGTATAG PS let-7d* (RFAM-M. musculus) 346965 1810ATCTGCACTGTCAGCACTTTA PS miR-106b (RFAM-M. musculus) 346966 1784ACATCGTTACCAGACAGTGTTA PS miR-200a (RFAM-M. musculus) 346967 1874GCGGAACTTAGCCACTGTGAA PS miR-27a (RFAM-M. musculus) 346968 1826CAGCTATGCCAGCATCTTGCCT PS miR-31 (RFAM-M. musculus) 346969 1829CAGGCCGGGACAAGTGCAATA PS miR-92 (RFAM-M. musculus) 346970 1849CTACCTGCACGAACAGCACTTTG PS miR-93 (RFAM-M. musculus) 346971 1801AGCAAAAATGTGCTAGTGCCAAA PS miR-96 (RFAM-M. musculus) 346972 1759AACAACCAGCTAAGACACTGCCA PS miR-172 (RFAM-M. musculus) 348169 1922TTCGCCCTCTCAACCCAGCTTTT PO miR-320 348170 1860 GAACCCACAATCCCTGGCTTA POmiR-321-1 348172 1908 TCCATAAAGTAGGAAACACTACA PO miR-142as (Michael etal) 348175 1905 TCATCATTACCAGGCAGTATTA PO miR-200b (Michael et al)348177 1820 CACAAATTCGGTTCTACAGGGTA PO miR-10b (Michael et al) 3481781878 GCTGGATGCAAACCTGCAAAACT PO miR-19b (Michael et al) 348180 1869GCAGAACTTAGCCACTGTGAA PO miR-27* (Michael et al) 348181 1858CTTCCAGTCAAGGATGTTTACA PO miR-97 (Michael et al) 348182 1855CTGGCTGTCAATTCATAGGTCA PO miR-192 (Michael et al) 348183 1922TTCGCCCTCTCAACCCAGCTTTT PS miR-320 348184 1860 GAACCCACAATCCCTGGCTTA PSmiR-321-1 348185 1886 GTAAACCATGATGTGCTGCTA PS miR-15b (Michael et al)348186 1908 TCCATAAAGTAGGAAACACTACA PS miR-142as (Michael et al) 3481881883 GGATTCCTGGGAAAACTGGAC PS miR-145 (Michael et al) 348189 1905TCATCATTACCAGGCAGTATTA PS miR-200b (Michael et al) 348190 1791ACTATACAATCTACTACCTCA PS let-7f (Michael et al) 348191 1820CACAAATTCGGTTCTACAGGGTA PS miR-10b (Michael et al) 348192 1878GCTGGATGCAAACCTGCAAAACT PS miR-19b (Michael et al) 348193 1873GCCTATCCTGGATTACTTGAA PS miR-26a (Michael et al) 348194 1869GCAGAACTTAGCCACTGTGAA PS miR-27* (Michael et al) 348195 1858CTTCCAGTCAAGGATGTTTACA PS miR-97 (Michael et al) 348196 1855CTGGCTGTCAATTCATAGGTCA PS miR-192 (Michael et al) 354168 1751AAACCACACAACCTACTACCTCA PS let-7b-Ruvkun 354169 1752AAACCATACAACCTACTACCTCA PS let-7c-Ruvkun 354170 1764AACTATGCAACCTACTACCTCT PS let-7d-Ruvkun 354171 1765AACTGTACAAACTACTACCTCA PS let-7gL-Ruvkun 354172 1760AACAGCACAAACTACTACCTCA PS let-7i-Ruvkun 354173 1924TTGGCATTCACCGCGTGCCTTAA PS mir-124a-Ruvkun 354174 1833CCAAGCTCAGACGGATCCGA PS mir-127-Ruvkun 354175 1896TACTTTCGGTTATCTAGCTTTA PS mir-131-Ruvkun 354176 1846CGGCCTGATTCACAACACCAGCT PS mir-138-Ruvkun 354177 1768ACAAACCATTATGTGCTGCTA PS mir-15-Ruvkun 354178 1789ACGCCAATATTTACGTGCTGCTA PS mir-16-Ruvkun 354179 1852CTATCTGCACTAGATGCACCTTA PS mir-18-Ruvkun 354180 1779ACAGCTGCTTTTGGGATTCCGTTG PS mir-191-Ruvkun 354181 1891TAACCGATTTCAGATGGTGCTA PS mir-29a-Ruvkun 354182 1813ATGCTTTGACAATACTATTGCACTG PS mir-301-Ruvkun 354183 1805AGCTGAGTGTAGGATGTTTACA PS mir-30b-Ruvkun 354184 1804AGCTGAGAGTGTAGGATGTTTACA PS mir-30c-Ruvkun 354185 1807AGCTTCCAGTCGGGGATGTTTACA PS mir-30d-Ruvkun 354186 1835CCAGCAGCACCTGGGGCAGTGG PS mir-324-3p-Ruvkun 354187 1899TATGGCAGACTGTGATTTGTTG PS mir-7-1*-Ruvkun 354188 1850CTACCTGCACTGTAAGCACTTTG PS mir-91-Ruvkun 354189 1822CACATAGGAATGAAAAGCCATA PS mir-135b (Ruvkun) 354190 1895TACTAGACTGTGAGCTCCTCGA PS mir-151* (Ruvkun) 354191 1885GGCTATAAAGTAACTGAGACGGA PS mir-340 (Ruvkun) 354192 1923TTCTAGGATAGGCCCAGGGGC PS mir-331 (Ruvkun) 354193 1892TACATACTTCTTTACATTCCA PS miR-1 (RFAM) 354194 1817 CAATCAGCTAACTACACTGCCTPS miR-34c (RFAM) 354195 1837 CCCCTATCACGATTAGCATTAA PS miR-155 (RFAM)354196 1910 TCCATCATTACCCGGCAGTATT PS miR-200c (RFAM) 354197 1818CAATCAGCTAATGACACTGCCT PS miR-34b (RFAM) 354198 1753AAACCCAGCAGACAATGTAGCT PS mir-221 (RFAM-M. musculus) 354199 1796AGACCCAGTAGCCAGATGTAGCT PS mir-222 (RFAM-M. musculus) 354200 1917TGAGCTCCTGGAGGACAGGGA PS mir-339-1 (RFAM) 354201 1925TTTAAGTGCTCATAATGCAGT PS miR-20* (human) 354202 1926TTTTCCCATGCCCTATACCTCT PS miR-202 (human) 354203 1856CTTCAGCTATCACAGTACTGTA PS miR-101b 354204 1894 TACCTGCACTGTTAGCACTTTG PSmiR-106a 354205 1772 ACAAGTGCCCTCACTGCAGT PS miR-17-3p 354206 1859GAACAGGTAGTCTAAACACTGGG PS miR-199b (mouse) 354207 1915TCTTCCCATGCGCTATACCTCT PS miR-202 (mouse) 354208 1808AGGCAAAGGATGACAAAGGGAA PS miR-211 (mouse) 354209 1809ATCCAGTCAGTTCCTGATGCAGTA PS miR-217 (mouse) 354210 1888TAAACGGAACCACTAGTGACTTA PS miR-224 (RFAM-mouse) 354211 1758AACAAAATCACAAGTCTTCCA PS miR-7b 354212 1919 TGTAAGTGCTCGTAATGCAGT PSmiR-20* (mouse) 354213 1778 ACACTTACTGGACACCTACTAGG PS mir-325 (human)354214 1777 ACACTTACTGAGCACCTACTAGG PS mir-325 (mouse) 354215 1877GCTGGAGGAAGGGCCCAGAGG PS mir-326 (human) 354216 1794ACTGGAGGAAGGGCCCAGAGG PS mir-326 (mouse) 354217 1755AAAGAGGTTAACCAGGTGTGTT PS mir-329-1 (human) 354218 1750AAAAAGGTTAGCTGGGTGTGTT PS mir-329-1 (mouse) 354219 1914TCTCTGCAGGCCGTGTGCTTTGC PS mir-330 (human) 354220 1913TCTCTGCAGGCCCTGTGCTTTGC PS mir-330 (mouse) 354221 1757AAAGGCATCATATAGGAGCTGGA PS mir-337 (human) 354222 1756AAAGGCATCATATAGGAGCTGAA PS mir-337 (mouse) 354223 1872GCCCTGGACTAGGAGTCAGCA PS mir-345 (human) 354224 1868GCACTGGACTAGGGGTCAGCA PS mir-345 (mouse) 354225 1799AGAGGCAGGCATGCGGGCAGACA PS mir-346 (human) 354226 1798AGAGGCAGGCACTCGGGCAGACA PS mir-346 (mouse) 354228 1841CCTCAAGGAGCCTCAGTCTAGT PS miR-151 (rat) 354229 1797AGAGGCAGGCACTCAGGCAGACA PS miR-346 (rat) 354230 1819CAATCAGCTAATTACACTGCCTA PS miR-34b (mouse) 354231 1842CCTCAAGGAGCTTCAGTCTAGT PS miR-151 (human) 354232 1751AAACCACACAACCTACTACCTCA PO let-7b-Ruvkun 354234 1764AACTATGCAACCTACTACCTCT PO let-7d-Ruvkun 354235 1765AACTGTACAAACTACTACCTCA PO let-7gL-Ruvkun 354236 1760AACAGCACAAACTACTACCTCA PO let-7i-Ruvkun 354238 1833 CCAAGCTCAGACGGATCCGAPO mir-127-Ruvkun 354239 1896 TACTTTCGGTTATCTAGCTTTA PO mir-131-Ruvkun354240 1846 CGGCCTGATTCACAACACCAGCT PO mir-138-Ruvkun 354242 1789ACGCCAATATTTACGTGCTGCTA PO mir-16-Ruvkun 354243 1852CTATCTGCACTAGATGCACCTTA PO mir-18-Ruvkun 354244 1779ACAGCTGCTTTTGGGATTCCGTTG PO mir-191-Ruvkun 354245 1891TAACCGATTTCAGATGGTGCTA PO mir-29a-Ruvkun 354246 1813ATGCTTTGACAATACTATTGCACTG PO mir-301-Ruvkun 354248 1804AGCTGAGAGTGTAGGATGTTTACA PO mir-30c-Ruvkun 354250 1835CCAGCAGCACCTGGGGCAGTGG PO mir-324-3p-Ruvkun 354251 1899TATGGCAGACTGTGATTTGTTG PO mir-7-1*-Ruvkun 354253 1822CACATAGGAATGAAAAGCCATA PO mir-135b (Ruvkun) 354254 1895TACTAGACTGTGAGCTCCTCGA PO mir-151* (Ruvkun) 354255 1885GGCTATAAAGTAACTGAGACGGA PO mir-340 (Ruvkun) 354256 1923TTCTAGGATAGGCCCAGGGGC PO mir-331 (Ruvkun) 354258 1817CAATCAGCTAACTACACTGCCT PO miR-34c (RFAM) 354259 1837CCCCTATCACGATTAGCATTAA PO miR-155 (RFAM) 354260 1910TCCATCATTACCCGGCAGTATT PO miR-200c (RFAM) 354261 1818CAATCAGCTAATGACACTGCCT PO miR-34b (RFAM) 354264 1917TGAGCTCCTGGAGGACAGGGA PO mir-339-1 (RFAM) 354265 1925TTTAAGTGCTCATAATGCAGT PO miR-20* (human) 354266 1926TTTTCCCATGCCCTATACCTCT PO miR-202 (human) 354267 1856CTTCAGCTATCACAGTACTGTA PO miR-101b 354268 1894 TACCTGCACTGTTAGCACTTTG POmiR-106a 354269 1772 ACAAGTGCCCTCACTGCAGT PO miR-17-3p 354270 1859GAACAGGTAGTCTAAACACTGGG PO miR-199b (mouse) 354271 1915TCTTCCCATGCGCTATACCTCT PO miR-202 (mouse) 354272 1808AGGCAAAGGATGACAAAGGGAA PO miR-211 (mouse) 354273 1809ATCCAGTCAGTTCCTGATGCAGTA PO miR-217 (mouse) 354274 1888TAAACGGAACCACTAGTGACTTA PO miR-224 (RFAM-mouse) 354275 1758AACAAAATCACAAGTCTTCCA PO miR-7b 354276 1919 TGTAAGTGCTCGTAATGCAGT POmiR-20* (mouse) 354277 1778 ACACTTACTGGACACCTACTAGG PO mir-325 (human)354278 1777 ACACTTACTGAGCACCTACTAGG PO mir-325 (mouse) 354279 1877GCTGGAGGAAGGGCCCAGAGG PO mir-326 (human) 354280 1794ACTGGAGGAAGGGCCCAGAGG PO mir-326 (mouse) 354281 1755AAAGAGGTTAACCAGGTGTGTT PO mir-329-1 (human) 354282 1750AAAAAGGTTAGCTGGGTGTGTT PO mir-329-1 (mouse) 354283 1914TCTCTGCAGGCCGTGTGCTTTGC PO mir-330 (human) 354284 1913TCTCTGCAGGCCCTGTGCTTTGC PO mir-330 (mouse) 354285 1757AAAGGCATCATATAGGAGCTGGA PO mir-337 (human) 354286 1756AAAGGCATCATATAGGAGCTGAA PO mir-337 (mouse) 354287 1872GCCCTGGACTAGGAGTCAGCA PO mir-345 (human) 354288 1868GCACTGGACTAGGGGTCAGCA PO mir-345 (mouse) 354289 1799AGAGGCAGGCATGCGGGCAGACA PO mir-346 (human) 354290 1798AGAGGCAGGCACTCGGGCAGACA PO mir-346 (mouse) 354292 1841CCTCAAGGAGCCTCAGTCTAGT PO miR-151 (rat) 354293 1797AGAGGCAGGCACTCAGGCAGACA PO miR-346 (rat) 354294 1819CAATCAGCTAATTACACTGCCTA PO miR-34b (mouse) 354295 1842CCTCAAGGAGCTTCAGTCTAGT PO miR-151 (human)

It is also understood that, although many of the oligomeric compoundslisted in Tables 64-66 have been designed to target or mimic aparticular miRNA from humans, for example, that oligomeric compound mayalso target or mimic other miRNAs from mammals, such as those fromrodent species, for example. It is also understood that these miRNAs andmimics can serve as the basis for several variations of nucleic acidoligomeric compounds, including compounds with chemical modificationssuch as uniform or chimeric 2′-MOE oligomeric compounds, as well as LNAsand PNAs; such oligomeric compounds are also within the scope of theinvention. One such non-limiting example is ISIS Number 351104(CTAGTGGTCCTAAACATTTCAC; SEQ ID NO: 296), which is a PNA oligomericcompound targeted to the human mir-203 miRNA.

Example 35 Targeting miRNAs in Introns and Exons

By mapping the coding sequences of miRNAs onto genomic contigs (whichsequence information is available from public databases, such as GenBankand Locus Link), and identifying loci at which other reported genecoding sequences also co-map, it was observed that miRNAs can be encodedwithin the exons or introns of other genes. The oligomeric compounds ofthe present invention can be designed to target introns and exons ofthese genes. For example, the oligomeric compounds of the presentinvention can be designed to target introns or exons of the genes listedin Table 67. More specifically, these oligomeric compounds can targetthe miRNAs encoded within the exons or introns of these genes listed inTable 67.

TABLE 67 Oligomeric compounds targeting miRNAs found within introns orexons SEQ ID Locus SEQ ISIS # NO: Locus containing miRNA ID NO 327873291 Ubiquitin protein ligase WWP2 containing mir- 1928 140 327874 292hypothetical protein FLJ13189 1929 327877 295 deleted in lymphocyticleukemia, 2 containing 1930 mir-16-1 and mir-15a-1 327877 295 SMC4(structural maintenance of chromosomes 4, 1931 yeast)-like 1 containingmir-16-3 and mir-15b 327879 297 heterogeneous nuclear ribonucleoproteinK 1932 containing mir-7-1 327879 297 pituitary gland specific factor 1acontaining 1933 mir-7-3 327881 299 R3H domain (binds single-strandednucleic 1934 acids) containing containing mir-128a 327882 300 proteintyrosine phosphatase, receptor type, N 1935 polypeptide 2 containingmir-153-2 327882 300 protein tyrosine phosphatase, receptor type, N 1936containing mir-153-1 327883 301 chromosome 9 ORF3 containing mir-23b,mir-24-2 1937 and mir-27b 327892 310 Transcriptional activator of thec-fos 1938 promoter containing mir-131-1/miR-9 327896 314 hypotheticalprotein MGC14376 containing mir- 1939 22 327906 324 hypothetical proteinFLJ11729 containing mir- 1940 103-2 327906 324 hypothetical proteinFLJ12899 containing mir- 1941 103-1 327907 325 conserved gene amplifiedin osteosarcoma 1942 containing miR-26a-2 327907 325 HYA22 proteincontaining miR-26a-1 1943 327908 326 Sterol regulatory element binding1944 transcription factor 2 containing mir-33a 327910 328 pantothenatekinase containing mir-107 1945 327912 330 upstream regulatory elementbinding protein 1 1946 containing mir-98 and let-7f-2 327915 333 slit(Drosophila) homolog 3 containing mir- 1947 218-2 327915 333 slit(Drosophila) homolog 2 containing mir- 1948 218-1 327923 341 cyclicAMP-regulated phosphoprotein, 21 kD 1949 containing mir-128b 327932 350transient receptor potential cation channel, 1950 subfamily M, member 3containing mir-204 327946 364 melastatin 1 containing mir-211 1951327947 365 RNA cyclase homolog containing mir-101-3 1952 327954 372CGI-120 protein containing mir-148b 1953 327963 381 nuclear LIMinteractor-interacting factor 1954 containing mir-26b 327964 382 COPZ2for nonclathrin coat protein zeta-COP 1955 containing mir-152 327967 385hypothetical protein PRO2730 containing let-7g 1956 327968 386 sterolregulatory element-binding protein-1/ 1957 mir-33b 328089 391 talin 2containing hypothetical miR-13/miR-190 1958 328091 393 calcitoninreceptor containing hypothetical 1959 miRNA 30 328092 394 glutamatereceptor, ionotrophic, AMPA 3/ 1960 hypothetical miRNA-033 328093 395myosin, heavy polypeptide 7B, cardiac muscle, 1961 beta containinghypothetical miRNA 039 328101 403 LOC 114614/hypothetical miRNA-071 1962328104 406 dachshund (Drosophila) homolog containing 1963 hypotheticalmiRNA 083 328105 407 DiGeorge syndrome critical region gene 8/ 1964hypothetical miRNA-088 328111 413 hypothetical protein FLJ21016,containing 1965 hypothetical miRNA 111 328117 419 collagen, type I,alpha 1/hypothetical miRNA- 1966 144 328119 421 hypothetical proteinHH114 containing 1967 hypothetical miRNA 154 328120 422 sprouty(Drosophila) homolog 4 containing 1968 hypothetical miRNA 156 328124 426ribosomal protein L5/hypothetical miRNA 168-2 1969 328125 427 forkheadbox P2/hypothetical miRNA 169 1970 328127 429 glutamate receptor,ionotropic, AMPA 2/ 1971 hypothetical miRNA 171 328128 430 potassiumlarge conductance calcium-activated 1972 channel, subfamily M, alphamember 1 containing hypothetical miRNA 172 328131 433 hypotheticalprotein FLJ20307 1973 328135 437 cezanne 2/hypothetical miRNA-180 1974328137 439 tight junction protein 1 (zona occludens 1)/ 1975hypothetical miRNA-183 340343 1780 gamma-aminobutyric acid (GABA) Areceptor, 1976 alpha 3 containing miR-105 (Mourelatos) and miR-105-2340348 848 Minichromosome maintenance deficient (S. cerevisiae) 1977 7containing miR-93 (Mourelatos) and miR-25 and miR-94 340350 855 KIAA1808protein containing miR-95 1978 (Mourelatos) 340356 1853 LIMdomain-containing preferred translocation 1979 partner in lipomacontaining miR-28 340360 1865 chromosome 9 open reading frame 5containing 1980 miR-32 341785 854 glypican 1 containing miR-149 1981341798 1871 Notch 4 like containing mir-123/mir-126 1982 341800 1766zinc finger protein 265 containing miR-186 1983 341801 1839follistatin-like 1 containing miR-198 1984 341802 1806 hypotheticalprotein FLJ10496 containing miR- 1985 191 341808 1861 hypotheticalprotein DKFZp761P1121, containing 1986 miR-185 341809 1786 chloridechannel 5 (nephrolithiasis 2, X- 1987 linked, Dent disease) containingmiR-188 341812 1771 myosin, heavy polypeptide 6, cardiac muscle, 1988alpha (cardiomyopathy, hypertrophic 1) containing miR-208 341813 938phosphodiesterase 2A, cGMP-stimulated 1989 containing miR-139 3446111785 mesoderm specific transcript (mouse) homolog 1990 containingmir-240* (Kosik) 344615 1900 Apoptosis-associated tyrosine kinase 1991containing mir-244* (Kosik) 344617 1862 RNB6 containing mir-248* (Kosik)1992 346692 1889 gamma-aminobutyric acid (GABA) A receptor, 1993epsilon, containing miR-224 (Sanger) 348128 1858 Nuclear transcriptionfactor Y, gamma 1994 containing miR-30c-2 and miR-30e

Example 36 Oligomeric Compounds Targeting Components of the RNAi Pathway

In one step of miRNA processing, the pre-miRNAs, approximately 70 to 110nucleotides in length, are processed by the human Dicer RNase intomature miRNAs. The Dicer enzyme is conserved from fungi to vertebrates.The helicase-moi gene is the human homolog of Dicer from Drosophila.Human Dicer is required for the production of active small non-codingRNAs involved in repressing gene expression by the RNA interferencepathway; targeted destruction in cultured human cells of the mRNAencoding human Dicer leads to accumulation of the let-7 pre-miRNA(Hutvagner, et al., 2001, Science 293(5531):834-8). Furthermore, thezebrafish Dicer1 ortholog was cloned and its expression disrupted bytarget-selected gene inactivation; in homozygous dicer1 mutants, aninitial build-up of miRNA levels produced by maternal Dicer1 wasobserved, but miRNA accumulation halted after a few days, and adevelopmental arrest was observed at around day 10, indicating thatmiRNA-producing Dicer1 is essential for vertebrate development(Wienholds, et al., 2003, Nat. Genet., 35(3):217-8). The Dicer gene hasalso been disrupted in mice. Loss of Dicer1 led to lethality early indevelopment, with Dicer1-null embryos depleted of stem cells. Coupledwith the inability to generate viable Dicer1-null embryonic stem cells,this suggests a role for Dicer and, by implication, the RNAi machineryin maintaining the stem cell population during early mouse development(Bernstein, et al., 2003, Nat. Genet., 35(3):215-7).

Thus, it was predicted that treatment of cells with oligomeric compoundstargeting human Dicer would result in an increase in expression levelsof miRNA precursor structures, and thus would be useful in increasingthe sensitivity of or enabling the detection of certain pre-miRNAsand/or pri-miRNAs otherwise beneath the limits of detection. It was alsopredicted that treatment of cells with oligomeric compounds targetinghuman Dicer would result in a decrease in mature miRNAs, leading todysregulation of miRNA-regulated targets. Thus, a transcriptomics- orproteomics-based approach could be used to compare and identify targetRNAs or proteins for which changes in expression levels correlatedirectly or inversely with the changes in mature miRNA levels. TargetRNAs or their downstream protein products which are being misregulatedupon treatment with oligomeric compounds targeting human Dicer, canthereby lead to the identification of any potential miRNA-regulatedtargets.

The present invention provides methods of maintaining a pluripotent stemcell comprising contacting the cell with an effective amount of anoligomeric compound targeting human Dicer. The pluripotent stem cell canbe present in a sample of cord blood or bone marrow, or may be presentas part of a cell line. In addition, the pluripotent stem cell can be anembryonic stem cell.

In some embodiments, oligomeric compounds ISIS Number 138648(GCTGACCTTTTTGCTTCTCA; herein incorporated as SEQ ID NO: 1995) and ISISNumber 138678 (CATAAACATTTCCATCAGTG; herein incorporated as SEQ ID NO:1996), both 5-10-52′-MOE gapmers with phosphorothioate backbones, weredesigned to target the human Dicer mRNA. These oligomeric compounds wereused to transfect the A549, T-24, HepG2, HMEC, T47D, HuVEC, and MCF7cell lines, as well as human primary dendritic cells, preadipocytes,differentiated adipocytes, and human spleen tissue, and the effects oftreatment with the oligomeric compounds on phenotypic parameters, suchas caspase activity and expression of markers of adipocytedifferentiation (aP2, HSL, Glut4) was assessed as described in Examples11 and 13, respectively.

Interestingly, treatment of T47D breast adenocarcinoma (p53 mutant)cells with the oligomeric compound ISIS 138648 targeting human Dicer wasobserved to result in a 41% increase in caspase activity. This phenotypeis similar to the effect of treatment of T47D cells with oligomericcompound ISIS Number 328645 (SEQ ID NO: 554), targeting mir-124a-1described in Example 11. It is believed that treatment of T47D cellswith the oligomeric compound ISIS 138648 inhibits expression of humanDicer, which results in reduced production of mature miRNAs. Inadequatelevels of miRNAs or inappropriately elevated levels of miRNA precursorsmay disrupt important cellular events, such as regulation of the cellcycle, and lead cells to trigger apoptotic pathways.

In adipocyte differentiation assays performed as described in Example13, treatment of human white preadipocytes with ISIS Number 138648targeting human Dicer was observed to result in decreased triglycerideproduction. An increase in triglyceride content is a well-establishedmarker of adipocyte differentiation; treatment of adipocytes witholigomeric compound ISIS 138648 resulting in a decrease in triglyceridelevels indicates an apparent inhibition of adipocyte differentiation.Thus, the oligomeric compound ISIS 138648 targeting human Dicer may beuseful as a pharmaceutical agent with applications in the treatment,attenuation or prevention of obesity, hyperlipidemia, atherosclerosis,atherogenesis, diabetes, hypertension, or other metabolic diseases aswell as in the maintenance of the undifferentiated, pluripotentphenotype of stem or precursor cells. The inhibition of expression ofhuman Dicer by ISIS 138648 is believed to result in decreased productionof miRNAs, and some of these miRNAs may be critical for properregulation of the cell cycle (such as is predicted for the regulation ofERK5 by mir-143); treatment of preadipocytes with this inhibitor ofhuman Dicer and the resulting decrease in production of mature miRNAs,as well as the concommitant accumulation of pre-miRNAs or pri-miRNAs mayupset the balance between cellular proliferation and differentiation,predisposing cells to an undifferentiated state.

Example 37 Design of Additional Double-Stranded miRNA Mimics

As described supra, a reporter vector system employing, for example, thepGL3-bulge(×3) plasmid or the pGL3-mir-143 sensor plasmids can be usedto assess the ability of miRNA mimics to bind target sites or to assesstheir effects on the expression of miRNAs, pre-miRNAs or pri-miRNAs.Various chemically modified miRNA mimics have been designed andsynthesized for this purpose. The oligomeric compounds of the presentinvention can be designed to mimic a pri-miRNA, pre-miRNA or a single-or double-stranded miRNA while incorporating certain chemicalmodifications that alter one or more properties of the mimic, thuscreating a construct with superior qualities over the endogenousprecursor or miRNA.

In accordance with the present invention, a series of oligomericcompounds was designed and synthesized to mimic double-stranded miRNAs.In some embodiments, various oligomeric compounds representing the sensestrand of the mir-143 miRNA, were synthesized, incorporating variouschemically modified sugars and/or internucleoside linkages. Similarly,various oligomeric compounds representing the antisense strandcomplementary to the mir-143 miRNA were synthesized, incorporatingvarious chemically modified sugars and/or internucleoside linkages. Theantisense and sense oligomeric compounds designed to mimic mir-143 areshown in Table 68 and 69, respectively. All of the sugar moieties of theoligomeric compounds listed in Tables 68 and 69 are ribonucleotidesunless otherwise indicated, and the 3′-terminal nucleosides each have a3′-OH group unless otherwise specified. The sequences are written in the5′ to 3′ direction. All antisense oligomeric compounds in Table 68 havethe nucleotide sequence GAGCUACAGUGUUCAUCUCA (herein incorporated as SEQID NO: 1864). The sense oligomeric compounds in Table 69 have one ofthree nucleotide sequences which only differ in that there is athymidine substitution in place of uridine in two of the sequences;these are: UGAGAUGAAGCACUGUAGCUC (herein incorporated as SEQ ID NO:1088), UGAGATGAAGCACUGUAGCUC (herein incorporated as SEQ ID NO: 1088),and UGAGAUGAAGCACUGTAGCUC (herein incorporated as SEQ ID NO: 1088). InTables 68 and 69, the column “Chemical modification” lists the generalclass and type of chemical modification for the respective oligomericcompounds. The column “Sequence” indicates the nucleobase sequence withsymbols indicating sugar and linkage modifications. In the Sequencecolumns of Tables 68 and 69, internucleoside linkages are assumed to bephosphodiester unless otherwise indicated; phosphorothioateinternucleoside linkages are indicated by “s” after the letterindicating the nucleobase (for example, “GsC” indicates a guanosinelinked to a cytidine with a 3′,5′-phosphorothioate (PS) internucleosidelinkage). Other symbols used to indicate sugar and linkage modificationsin the Sequence columns of Tables 68 and 69 are as follows: “mc”indicates that the cytidine residue at the specified position is a5-methylcytidine; replacement of the 2′-OH of the ribosyl sugar with a2′-O-methoxyethyl (2′-MOE) is indicated by “e” after the letterindicating the nucleobase (for example, “GAe” indicates a guanosinelinked to a 2′-MOE adenosine with a 3′,5′-phosphodiester internucleosidelinkage); replacement or substitution of the 2′-OH of the ribosyl sugarwith a 2-O-methyl (2′-OMe) is indicated by “m” after the letterindicating the nucleobase (for example, “CmA” indicates a 2′-O-methylcytidine linked to an adenosine with a 3′,5′-phosphodiesterinternucleoside linkage); nucleosides having a 2′-Fluoro (2′-F)substituent group are indicated with a “f” after the letter indicatingthe nucleobase (for example, “GfAm” indicates a 2′-F guanosine linked toa 2′-O-Methyl-adenosine with a 3′,5′-phosphodiester internucleosidelinkage); 4′-Thio (4′-S) residues are indicated by “4s” (for example,“GC4s” indicates a guanosine linked to a 4′-S cytidine with a3′,5′-phosphodiester internucleoside linkage).

In the “Chemical modification” column of Tables 68 and 69, “unmodified”indicates a native strand. “Full” indicates a fully modified oligomericcompound where the chemical modification occurs at each nucleoside orinternucleoside linkage. For example each nucleoside of the oligomericcompound could have a modified sugar selected from one of 4′-S, 2′-MOE,2′-F, 2′-O-Methyl, LNA or ENA™ or could have uniformly modifiedinternucleoside linkages such as uniform phosphorothioateinternucleoside linkages.

In the “Chemical modification” column of Tables 68 and 69, “Alt”indicates that the nucleosides and or the internucleoside linkages havean alternating motif. The alternating motif can be the result ofdifferent sugar modifications that alternate (for example, 2′-ribosealternating with a 2′-modification other than ribose such as MOE, 2′-For 2′-O-Methyl, or alternating fully modified sugars such as 2′-O-Methylalternating with 2′-F), or can be the result of alternatinginternucleoside linkages (for example alternating phosphodiester andphosphorothioate internucleoside linkages). Oligomeric compounds havingalternating modifications are described in the chemical modificationcolumn with the modification at the first 5′-nucleoside or the firstinternucleoside linkage at the 5′-end of the nucleoside listed first.For example, oligomeric compounds described as “Alt 2′-F/2′-OMe” have a2′-F modified sugar at the 5′-terminal nucleoside with the nextnucleoside having a 2′-F modified sugar and this alternating pattern isrepeated through to the 3′-terminal nucleoside.

In the “Chemical modification” column of Tables 68 and 69, “gapmer”indicates that the oligomeric compound is divided into three distinctregions. The wings are the regions located externally at the 3′ and the5′-end with the gap being the internal region. Gapmers can be the resultof differences in linkage (PO vs. PS) or nucleoside (modified sugarmoiety or heterocyclic base). Gapmers also include chimeric gappedoligomeric compounds such as when the wings and the gapped regions areall distinct one from each other. Examples of chemistries that can beused to prepare gapped oligomeric compounds include 2′-MOE, 2′-F,2′-O-Methyl, LNA and ENA™.

In the “Chemical modification” column of Tables 68 and 69, “hemimer”indicates an oligomeric compound that has two distinct regions resultingfrom differences in the nucleoside or the internucleoside linkage orboth. Examples include oligomeric compounds having two regions whereinone region has modified internucleoside linkages such as PS or modifiedsugar moieties such as 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENA™.

In the “Chemical modification” column of Tables 68 and 69, “blockmer”indicates an oligomeric compound that has at least one block of modifiednucleosides or internucleoside linkages that are located internally. Theblocks are generally from two to about five nucleosides in length andare not located at one of the ends as that could be a hemimer. Examplesof blockmers include oligomeric compounds having from two to about fiveinternally modified nucleosides such as 2′-MOE, 2′-F, 2′-O-Methyl, LNAand ENA™.

In the “Chemical modification” column of Tables 68 and 69, “pointmodification” indicates an oligomeric compound having a single modifiednucleoside located in the oligomeric compound at any position.

TABLE 68 Antisense oligomeric compounds mimicking mir-143 SEQ ISIS IDChemical NO: NO modification Sequence 348173 1864 UnmodifiedGAGCUACAGUGCUUCAUCUCA 348187 1864 Full PS GsAsGsCsUsAsCsAsGsUsGsCsUsUsCsAsUsCsUsCsA 362972 1864 Alt ribose/2′- GAeGCeUAeCAeGUeGCeUUeCA MOEeUCeUCeA 366179 1864 Alt ribose/2′- GAmGCmUAmCAmGUmGCmUUmCAm OMe UCmUCmA366181 1864 Alt 2′- GmAGmCUmACmAGmUGmCUmUCmA OMe/ribose UmCUmCAm 3661821864 Full 2′-OMe GmAmGmCmUmAmCmAmGmUmGm CmUmUmCmAmUmCmUmCmAm 366188 18642′-MOE 3-15-3 GeAeGeCUACAGUGCUUCAUCUeC gapmer eAe 366189 1864 Full2′-MOE GeAeGeCeUeAeCeAeGeUeGeCe UeUeCeAeUeCeUeCeAe 366190 1864 Alt 2′-GeAGeCUeACeAGeUGeCUeUCeA MOE/ribose UeCUeCAe 366198 1864 Alt 2′-F/2′-GfAmGfCmUfAmCfAmGfUmGfCm OMe UfUmCfAmUfCmUfCmAf

TABLE 69 Sense oligomeric compounds mimicking mir-143 SEQ ISIS IDChemical NO: NO modification Sequence 348201 1088 UnmodifiedUGAGAUGAAGCACUGUAGCUC 342199 220 Unmodified UGAGAUGAAGCACUGUAGCUCA348215 1088 Full PS UsGsAsGsAsUsGsAsAsGsCsAsCsUsGsUsAsGsCsUsC 3661751088 PO/PS/PO UGAGAUGAAGsCsAsCsUsGUAGCUC gapmer 366176 1088 5′ PShemimer UsGsAsGsAsUGAAGCACUGUAGCUC 366177 1088 3′ PS hemimerUGAGAUGAAGCACUGUsAsGsCsUsC 366178 1088 Alt 2′-UmGAmGAmUGmAAmGCmACmUGmUAmGCmUCm OMe/ribose 366180 1088 AltUGmAGmAUmGAmAGmCAmCUmGUmAGmCUmC ribose/2′-OMe 366183 1088 2′-OMeUGAGAUmGmAAGmCmACUGUAGCmUmCm blockmer 366184 1088 2′-OMeUGAGAUGAmAmGCAmCmUGUAGCmUmCm blockmer 366185 1088 2′-MOEUGAGAUGAAGCAeCeUGUAGCUC blockmer 366186 1088 2′-MOEUGAGeAeUeGAAGCACUGUAGCUC blockmer 366187 1088 2′-MOEUGAGAUGAAGCACUGUeAeGeCUC blockmer 366191 1088 4′s gapmerU4sGAGAUGAAGCACUGUAGC4sU4sC4s 366192 1088 4′s 2′-OMeU4sGAGAUGAAGCACUGUAGCmUmCm gapmer 366193 1088 2′-F blockmerUGfAfGfAfUfGfAfAfGCACUGUAGCUC 366194 1088 LNA blockmerUGAGlAlUlGAAGCACUGUAGCUC 366195 1088 LNA blockmerUGAGAUGAAGCACUGUlAlGlCUC 366196 1088 LNA blockmerUGAGAUGAAGCAlClUGUAGCUC 366197 1088 Alt 2′-UmGfAmGfAmUfGmAfAmGfCmAfCmUfGmUfAmGfCmUfCm OMe/2′-F 366209 1088 LNAblockmer UGAGlAlTlGAAGCACUGUAGCUC 366210 1088 LNA blockmerUGAGAUGAAGCACUGTlAlGlCUC 366211 1088 LNA pointUGAGAUGAAGCAl^(m)ClUGUAGCUC modification

Oligomeric compounds representing mimics of the antisense and the sensestrands of a double-stranded miRNA can be hybridized, and variouscombinations of synthetic, modified or unmodified double-strandedoligomeric compounds, each representing a double-stranded miRNA mimic,may be formed. With the various chemical modifications, manypermutations of such double-stranded miRNA mimics can be achieved. Thesedouble-stranded oligomeric compounds can be blunt-ended or can comprisetwo strands differing in length such that the resulting double-strandedoligomeric compound has a 3′- and/or a 5′-overhang of one to fivenucleotides on either the sense and/or antisense strands. The compoundscan be analyzed for their ability to mimic miRNAs, pre-miRNAs, orpri-miRNAs and to bind to nucleic acid targets (for example, RNAtranscripts, mRNAs, reporter contructs), for their effects on miRNA,pre-miRNA, or pri-miRNA expression levels by quantitative real-time PCR,or they can be used in other in vivo or in vitro phenotypic assays toinvestigate the role of miRNAs in regulation of downstream nucleic acidtargets, as described in other examples herein. These oligomericcompounds of the present invention may disrupt pri-miRNA and/orpre-miRNA structures, and sterically hinder cleavage by Drosha-likeand/or Dicer-like Rnase III enzymes, respectively. Oligomeric compoundscapable of binding to the mature miRNA are also predicted to prevent theRISC-mediated binding of a miRNA to its mRNA target, either by cleavageor steric occlusion of the miRNA.

In some embodiments, HeLa cells transiently expressing the pGL3-mir-143sensor reporter vector and the pRL-CMV Renilla luciferase plasmids, asdescribed in Example 27, were also treated with double-strandedoligomeric compounds produced by hybridizing an antisense oligomericcompound from Table 68 with a sense oligomeric compound from Table 69,as described herein. HeLa cells were routinely cultured and passaged andon the day before transfection, the HeLa cells were seeded onto 96-wellplates 3,000 cells/well. Cells were transfected according to standardpublished procedures with plasmids using 2 μg Lipofectamine™ 2000Reagent (Invitrogen) per μg of plasmid DNA, or, when transfectingdouble-stranded oligomeric compounds, 1.25 μg of Lipofectamine™ 2000Reagent was used per 100 nM oligonucleotide. Cells were treated at 10 nMand 100 nM with the double-stranded oligomeric compound mimics. Adouble-stranded oligomeric compound representing a 10-base mismatchedsequence antisense to the unrelated PTP1B mRNA, composed of ISIS Number342427 (SEQ ID NO: 863) hybridized to its perfect complement ISIS Number342430 (SEQ ID NO: 864) was used as a negative control (“ds-Control”).The pGL3-mir-143 sensor reporter plasmid was transfected at 0.025 μg perwell. The luciferase signal in each well was normalized to the Renillaluciferase (RL) activity produced from the co-transfected pRL-CMVplasmid, which was transfected at 2.5 μg per well. In accordance withmethods described in Example 12 and 27, a luciferase assay was performed48-hours after transfection. Briefly, cells were lysed in passive lysisbuffer (PLB; Promega), and 20 ul of the lysate was then assayed for RLactivity using a Dual Luciferase Assay kit (Promega) according to themanufacturer's protocol. The results below are an average of two trialsand are presented as percent pGL3-Control luciferase expressionnormalized to pRL-CMV expression (RL). The data are shown in Table 70.

TABLE 70 Luciferase assays showing effects of double-stranded compoundsmimicking mir-143 ISIS Numbers luciferase expression hybridized to formds (% lucif.only control) compound 10 nM treatment 100 nM treatmentpGL3-mir-143 sensor + 79.4 94.1 pRL-CMV only pGL3-mir-143 sensor + 120.6105.9 pRL-CMV only 342430 + 342427 75.0 86.1 ds-Control 348215 + 34817323.1 37.5 348215 + 362972 28.6 32.4 366175 + 348173 20.0 25.0 366175 +362972 56.9 33.4 366176 + 348173 42.6 30.0 366176 + 362972 63.4 98.5366177 + 348173 35.7 33.6 366177 + 362972 32.8 29.1 366183 + 348173 29.224.5 366183 + 362972 54.3 36.8 366184 + 348173 35.6 27.7 366184 + 36297247.3 31.9 366185 + 348173 22.2 18.5 366185 + 362972 27.2 28.7 366186 +348173 34.8 26.8 366186 + 362972 50.2 60.8 366187 + 348173 34.6 32.4366187 + 362972 25.5 27.9 366209 + 348173 112.9 85.4 366209 + 362972111.3 97.5 366210 + 348173 37.1 28.2 366210 + 362972 51.8 41.1 366211 +348173 32.1 28.7 366211 + 362972 46.6 36.7 366193 + 348173 20.0 17.6366193 + 362972 24.4 22.6 366191 + 348173 27.3 26.9 366191 + 362972 37.525.8 366192 + 348173 22.3 27.9 366192 + 362972 28.9 25.7 366197 + 34817337.0 22.2 366197 + 362972 42.0 32.7 366197 + 366198 30.2 28.7 366178 +348173 75.0 74.0 366178 + 362972 98.6 104.0 366178 + 366179 63.5 75.4366178 + 366181 74.1 70.6 366180 + 366179 97.0 38.5 366180 + 366181 43.550.2 pGL3-mir-143 sensor + 100.0 112.9 pRL-CMV only 342430 + 342427 81.2165.9 ds-Control 348201 + 348187 44.0 55.4 348201 + 366182 138.9 89.2348201 + 366179 76.2 68.5 348201 + 366181 92.2 340.0 348201 + 36297265.2 67.3 348201 + 366198 47.3 58.8 342199 + 348173 40.3 122.0 342199 +348187 91.3 55.5 342199 + 366182 47.4 84.1 342199 + 366179 76.5 45.9342199 + 366181 86.1 34.2 342199 + 362972 50.8 78.7 342199 + 366189 26.745.2 342199 + 366190 93.0 37.9 342199 + 366198 52.5 45.5

From these data, it was observed that treatment of HeLa cells expressingthe pGL3-mir-143 sensor reporter vector with many of the double-strandedoligomeric compounds mimicking mir-143 at both the 10 nM and 100 nMconcentrations resulted in inhibition of luciferase activity. Forexample, the double stranded oligomeric compounds comprising ISIS Number348173 as an unmodified antisense strand in combination with ISIS Number366177 (a hemimer with phosphorothioate modified residues at the 3′ end)or ISIS Number 366185 (a 2′-MOE blockmer) as the modified sense strandresulted in significant reductions in luciferase activity. Furthermore,double stranded oligomeric compounds comprising, as the antisensestrand, either ISIS Number 366189 (a fully modified 2′-MOE compound) orISIS Number 366198 (with alternating 2′-Fluoro and 2′-O-Methylresidues), in combination with ISIS Number 342199 as the unmodifiedsense strand resulted in significant reductions in luciferase activity,indicating that these compounds are effective mir-143 mimics. Taken withthe previous observations that the mir-143 miRNA is involved inadipocyte differentiation, these double-stranded mir-143 mimics may beuseful as therapeutic agents with applications in the treatment,attenuation or prevention of obesity, hyperlipidemia, atherosclerosis,atherogenesis, diabetes, hypertension, or other metabolic diseases aswell as having potential applications in the maintenance of thepluripotent phenotype of stem or precursor cells.

Example 38 Design of Oligomeric Compounds Targeting pri-miRNAs

As described above, mature miRNAs originate from pri-miRNAs, which arebelieved to be processed into pre-miRNAs by the Drosha RNase III enzyme,and subsequently exported from the nucleus to the cytoplasm, where thepre-miRNAs are processed by human Dicer into double-strandedintermediates resembling siRNAs, which are then processed into maturemiRNAs.

Some oligomeric compounds of the present invention are believed to bindto pri-miRNA molecules and interfere with their processing into a maturemiRNA. These oligomeric compounds were observed to affect a decrease inexpression levels of mature miRNA, presumably due, at least in part, tosteric interference with their processing into mature miRNAs by humanDicer. Furthermore, as described above, some oligomeric compounds of thepresent invention have been observed to affect an increase in expressionlevels of pri-miRNAs; it is believed that the decrease in levels ofmature miRNAs cells treated with these oligomeric compounds may triggera feedback mechanism that signals these cells to increase production ofthe pri-miRNA molecule. This increase may be the result, at least inpart, of a stimulation of transcription of the pri-miRNAs in response tothe decrease in mature miRNAs. Not mutually exclusive with theprocessing interference and the feedback mechanisms is the possibilitythat treatment with oligomeric compounds could stimulate the activity ofan RNA-dependent RNA polymerase (RdRP) that amplifies pre-miRNAs orpri-miRNAs.

In one embodiment, several nested series of single-stranded oligomericcompounds, 15-nucleotides in length, composed of 2′-methoxyethoxy(2′-MOE) modified nucleotides and phosphorothioate (P═S) internucleosidelinkages throughout the compound, were designed and synthesized totarget several pri-miRNAs, to test the effects of these compounds on theexpression levels of small non-coding RNAs. These compounds are shown inTable 71, below. “Pri-miRNA” indicates the particular pri-miRNA whichcontains the miRNA that the oligomeric compound was designed to target.The “Region” column describes the general region of the pri-miRNA thatis being targeted. The following features of the stemloop structures ofpri-miRNA were targeted: 1) “5′-stem side mir start” means the 5′-stemside at the 5′-end of the sequence representing the mature miRNA, withthe oligomeric compounds targeting and spanning sequences completelyoutside of the mature miRNA to completely within it; 2) “5′-stem sidemir end” means the 5′-stem side at the 3′-end of the sequencerepresenting the mature miRNA, with the oligomeric compounds targetingand spanning sequences completely within the mature miRNA to spanningand extending beyond the 3′-end of it; 3) “loop start” means the 5′-sideof the loop region; 4) “loop end” means with the oligomeric compoundstargeting and ending at the 3′-side of the loop region; 5) “3′-stem sidemir start” means the 3′-stem side at the 5′-end of the sequencerepresenting the mature miRNA, with the oligomeric compounds targetingand completely within the mature miRNA to a few nucleotides outside ofit; 6) “3′-stem side mir end” means the 3′-stem side at the 3′-end ofthe sequence representing the mature miRNA, with the oligomericcompounds targeting and spanning sequences completely within the maturemiRNA to completely outside of it.

TABLE 71 Uniform 2′-MOE oligomeric compounds targeting pri-miRNAs SEQ IDpri-miRNA Region Isis # Sequence NO: mir-182 mir-182 5′-stem side mirstart 366888 AAACGGGGGGAGGCA 1997 mir-182 mir-182 5′-stem side mir start366889 GCCAAAAACGGGGGG 1998 mir-182 mir-182 5′-stem side mir start366890 ATTGCCAAAAACGGG 1999 mir-182 mir-182 5′-stem side mir start366891 ACCATTGCCAAAAAC 2000 mir-182 mir-182 5′-stem side mir start366892 TCTACCATTGCCAAA 2001 mir-182 mir-182 5′-stem side mir end 366893TGTGAGTTCTACCAT 2002 mir-182 mir-182 5′-stem side mir end 366894CAGTGTGAGTTCTAC 2003 mir-182 mir-182 5′-stem side mir end 366895CACCAGTGTGAGTTC 2004 mir-182 mir-182 5′-stem side mir end 366896CCTCACCAGTGTGAG 2005 mir-182 mir-182 loop start 366897 TCCTGTTACCTCACC2006 mir-182 mir-182 loop start 366898 GATCCTGTTACCTCA 2007 mir-182mir-182 loop start 366899 CGGATCCTGTTACCT 2008 mir-182 mir-182 loop end366900 TGTTACCTCACCAGT 2009 mir-182 mir-182 loop end 366901CCTGTTACCTCACCA 2010 mir-182 mir-182 loop end 366902 ATCCTGTTACCTCAC2011 mir-182 mir-182 loop end 366903 GGATCCTGTTACCTC 2012 mir-182mir-182 loop end 366904 CCGGATCCTGTTACC 2013 mir-182 mir-182 3′-stemside mir start 366905 GAACCACCGGATCCT 2014 mir-182 mir-182 3′-stem sidemir start 366906 CTAGAACCACCGGAT 2015 mir-182 mir-182 3′-stem side mirstart 366907 AGTCTAGAACCACCG 2016 mir-182 mir-182 3′-stem side mir start366908 GCAAGTCTAGAACCA 2017 mir-182 mir-182 3′-stem side mir end 366909ATAGTTGGCAAGTCT 2018 mir-182 mir-182 3′-stem side mir end 366910CGCCCCATAGTTGGC 2019 mir-182 mir-182 3′-stem side mir end 366911CCTCGCCCCATAGTT 2020 mir-182 mir-182 3′-stem side mir end 366912AGTCCTCGCCCCATA 2021 mir-182 mir-182 3′-stem side mir end 366913CTGAGTCCTCGCCCC 2022 mir-216 mir-216 5′-stem side mir start 366914AAGCCAACTCACAGC 2023 mir-216 mir-216 5′-stem side mir start 366915AGATTAAGCCAACTC 2024 mir-216 mir-216 5′-stem side mir start 366916CTGAGATTAAGCCAA 2025 mir-216 mir-216 5′-stem side mir start 366917CAGCTGAGATTAAGC 2026 mir-216 mir-216 5′-stem side mir start 366918TGCCAGCTGAGATTA 2027 mir-216 mir-216 5′-stem side mir end 366919TCACAGTTGCCAGCT 2028 mir-216 mir-216 5′-stem side mir end 366920ATCTCACAGTTGCCA 2029 mir-216 mir-216 5′-stem side mir end 366921AACATCTCACAGTTG 2030 mir-216 mir-216 5′-stem side mir end 366922ATGAACATCTCACAG 2031 mir-216 mir-216 loop start 366923 ATTGTATGAACATCT2032 mir-216 mir-216 loop start 366924 GGATTGTATGAACAT 2033 mir-216mir-216 loop start 366925 AGGGATTGTATGAAC 2034 mir-216 mir-216 loop end366926 TGTATGAACATCTCA 2035 mir-216 mir-216 loop end 366927TGAGGGATTGTATGA 2036 mir-216 mir-216 3′-stem side mir start 366928ACTGTGAGGGATTGT 2037 mir-216 mir-216 3′-stem side mir start 366929ACCACTGTGAGGGAT 2038 mir-216 mir-216 3′-stem side mir start 366930GAGACCACTGTGAGG 2039 mir-216 mir-216 3′-stem side mir start 366931CCAGAGACCACTGTG 2040 mir-216 mir-216 3′-stem side mir end 366932CATAATCCCAGAGAC 2041 mir-216 mir-216 3′-stem side mir end 366933GTTTAGCATAATCCC 2042 mir-216 mir-216 3′-stem side mir end 366934TCTGTTTAGCATAAT 2043 mir-216 mir-216 3′-stem side mir end 366935TGCTCTGTTTAGCAT 2044 mir-216 mir-216 3′-stem side mir end 366936AATTGCTCTGTTTAG 2045 mir-143 mir-143 5′-stem side mir start 366937AGGCTGGGAGACAGG 2046 mir-143 mir-143 5′-stem side mir start 366938ACCTCAGGCTGGGAG 2047 mir-143 mir-143 5′-stem side mir start 366939TGCACCTCAGGCTGG 2048 mir-143 mir-143 5′-stem side mir start 366940CACTGCACCTCAGGC 2049 mir-143 mir-143 5′-stem side mir start 366941CAGCACTGCACCTCA 2050 mir-143 mir-143 5′-stem side mir end 366942AGAGATGCAGCACTG 2051 mir-143 mir-143 5′-stem side mir end 366943ACCAGAGATGCAGCA 2052 mir-143 mir-143 5′-stem side mir end 366944CTGACCAGAGATGCA 2053 mir-143 mir-143 5′-stem side mir end 366945CAACTGACCAGAGAT 2054 mir-143 mir-143 loop start 366946 CAGACTCCCAACTGA2055 mir-143 mir-143 loop start 366947 CTCAGACTCCCAACT 2056 mir-143mir-143 loop start 366948 ATCTCAGACTCCCAA 2057 mir-143 mir-143 loop end366949 AACTGACCAGAGATG 2058 mir-143 mir-143 loop end 366950CCAACTGACCAGAGA 2059 mir-143 mir-143 loop end 366951 TCCCAACTGACCAGA2060 mir-143 mir-143 loop end 366952 ACTCCCAACTGACCA 2061 mir-143mir-143 3′-stem side mir start 366953 TTCATCTCAGACTCC 2062 mir-143mir-143 3′-stem side mir start 366954 TGCTTCATCTCAGAC 2063 mir-143mir-143 3′-stem side mir start 366955 CAGTGCTTCATCTCA 2064 mir-143mir-143 3′-stem side mir end 366956 TGAGCTACAGTGCTT 2065 mir-143 mir-1433′-stem side mir end 366957 TCTTCCTGAGCTACA 2066 mir-143 mir-143 3′-stemside mir end 366958 CTCTCTTCCTGAGCT 2067 mir-143 mir-143 3′-stem sidemir end 366959 CTTCTCTCTTCCTGA 2068 mir-143 mir-143 3′-stem side mir end366960 CAACTTCTCTCTTCC 2069 mir-23b mir-23b 5′-stem side mir start366961 AGCAGCCAGAGCACC 2070 mir-23b mir-23b 5′-stem side mir start366962 ACCCAAGCAGCCAGA 2071 mir-23b mir-23b 5′-stem side mir start366963 GGAACCCAAGCAGCC 2072 mir-23b mir-23b 5′-stem side mir start366964 CCAGGAACCCAAGCA 2073 mir-23b mir-23b 5′-stem side mir start366965 ATGCCAGGAACCCAA 2074 mir-23b mir-23b 5′-stem side mir end 366966AATCAGCATGCCAGG 2075 mir-23b mir-23b 5′-stem side mir end 366967ACAAATCAGCATGCC 2076 mir-23b mir-23b 5′-stem side mir end 366968GTCACAAATCAGCAT 2077 mir-23b mir-23b 5′-stem side mir end 366969TAAGTCACAAATCAG 2078 mir-23b mir-23b loop start 366970 AATCTTAAGTCACAA2079 mir-23b mir-23b loop start 366971 TTAATCTTAAGTCAC 2080 mir-23bmir-23b loop start 366972 TTTTAATCTTAAGTC 2081 mir-23b mir-23b loop end366973 CTTAAGTCACAAATC 2082 mir-23b mir-23b loop end 366974ATCTTAAGTCACAAA 2083 mir-23b mir-23b loop end 366975 TAATCTTAAGTCACA2084 mir-23b mir-23b loop end 366976 TTTAATCTTAAGTCA 2085 mir-23bmir-23b loop end 366977 ATTTTAATCTTAAGT 2086 mir-23b mir-23b 3′-stemside mir start 366978 TGTGATTTTAATCTT 2087 mir-23b mir-23b 3′-stem sidemir start 366979 CAATGTGATTTTAAT 2088 mir-23b mir-23b 3′-stem side mirstart 366980 TGGCAATGTGATTTT 2089 mir-23b mir-23b 3′-stem side mir start366981 CCCTGGCAATGTGAT 2090 mir-23b mir-23b 3′-stem side mir end 366982TGGTAATCCCTGGCA 2091 mir-23b mir-23b 3′-stem side mir end 366983GTTGCGTGGTAATCC 2092 mir-23b mir-23b 3′-stem side mir end 366984GTGGTTGCGTGGTAA 2093 mir-23b mir-23b 3′-stem side mir end 366985GTCGTGGTTGCGTGG 2094 mir-23b mir-23b 3′-stem side mir end 366986AAGGTCGTGGTTGCG 2095 mir-203 mir-203 5′-stem side mir start 366987GACCCAGCGCGCGAG 2096 mir-203 mir-203 5′-stem side mir start 366988CACTGGACCCAGCGC 2097 mir-203 mir-203 5′-stem side mir start 366989AACCACTGGACCCAG 2098 mir-203 mir-203 5′-stem side mir start 366990AAGAACCACTGGACC 2099 mir-203 mir-203 5′-stem side mir start 366991GTTAAGAACCACTGG 2100 mir-203 mir-203 5′-stem side mir end 366992TTGAACTGTTAAGAA 2101 mir-203 mir-203 5′-stem side mir end 366993CTGTTGAACTGTTAA 2102 mir-203 mir-203 5′-stem side mir end 366994GAACTGTTGAACTGT 2103 mir-203 mir-203 5′-stem side mir end 366995ACAGAACTGTTGAAC 2104 mir-203 mir-203 loop start 366996 AATTGCGCTACAGAA2105 mir-203 mir-203 loop start 366997 ACAATTGCGCTACAG 2106 mir-203mir-203 loop start 366998 TCACAATTGCGCTAC 2107 mir-203 mir-203 loop end366999 TACAGAACTGTTGAA 2108 mir-203 mir-203 loop end 367000GCTACAGAACTGTTG 2109 mir-203 mir-203 loop end 367001 GCGCTACAGAACTGT2110 mir-203 mir-203 loop end 367002 TTGCGCTACAGAACT 2111 mir-203mir-203 3′-stem side mir start 367003 TTTCACAATTGCGCT 2112 mir-203mir-203 3′-stem side mir start 367004 ACATTTCACAATTGC 2113 mir-203mir-203 3′-stem side mir start 367005 TAAACATTTCACAAT 2114 mir-203mir-203 3′-stem side mir start 367006 TCCTAAACATTTCAC 2115 mir-203mir-203 3′-stem side mir end 367007 CTAGTGGTCCTAAAC 2116 mir-203 mir-2033′-stem side mir end 367008 CCGGGTCTAGTGGTC 2117 mir-203 mir-203 3′-stemside mir end 367009 CCGCCGGGTCTAGTG 2118 mir-203 mir-203 3′-stem sidemir end 367010 CGCCCGCCGGGTCTA 2119 mir-203 mir-203 3′-stem side mir end367011 CCGCGCCCGCCGGGT 2120 mir-21 mir-21 5′-stem side mir start 367012GCTACCCGACAAGGT 2121 mir-21 mir-21 5′-stem side mir start 367013AAGCTACCCGACAAG 2122 mir-21 mir-21 5′-stem side mir start 367014GATAAGCTACCCGAC 2123 mir-21 mir-21 5′-stem side mir start 367015TCTGATAAGCTACCC 2124 mir-21 mir-21 5′-stem side mir start 367016CAGTCTGATAAGCTA 2125 mir-21 mir-21 5′-stem side mir end 367017TCAACATCAGTCTGA 2126 mir-21 mir-21 5′-stem side mir end 367018CAGTCAACATCAGTC 2127 mir-21 mir-21 5′-stem side mir end 367019CAACAGTCAACATCA 2128 mir-21 mir-21 5′-stem side mir end 367020ATTCAACAGTCAACA 2129 mir-21 mir-21 loop start 367021 GCCATGAGATTCAAC2130 mir-21 mir-21 loop start 367022 TTGCCATGAGATTCA 2131 mir-21 mir-21loop start 367023 TGTTGCCATGAGATT 2132 mir-21 mir-21 loop end 367024TTCAACAGTCAACAT 2133 mir-21 mir-21 loop end 367025 GATTCAACAGTCAAC 2134mir-21 mir-21 loop end 367026 GAGATTCAACAGTCA 2135 mir-21 mir-21 loopend 367027 ATGAGATTCAACAGT 2136 mir-21 mir-21 loop end 367028CCATGAGATTCAACA 2137 mir-21 mir-21 3′-stem side mir start 367029GTGTTGCCATGAGAT 2138 mir-21 mir-21 3′-stem side mir start 367030CTGGTGTTGCCATGA 2139 mir-21 mir-21 3′-stem side mir start 367031CGACTGGTGTTGCCA 2140 mir-21 mir-21 3′-stem side mir start 367032CATCGACTGGTGTTG 2141 mir-21 mir-21 3′-stem side mir end 367033GACAGCCCATCGACT 2142 mir-21 mir-21 3′-stem side mir end 367034ATGTCAGACAGCCCA 2143 mir-21 mir-21 3′-stem side mir end 367035AAATGTCAGACAGCC 2144 mir-21 mir-21 3′-stem side mir end 367036CAAAATGTCAGACAG 2145 mir-221 mir-221 5′-stem side mir start 367037CATGCCCCAGACCTG 2146 mir-221 mir-221 5′-stem side mir start 367038AGGTTCATGCCCCAG 2147 mir-221 mir-221 5′-stem side mir start 367039GCCAGGTTCATGCCC 2148 mir-221 mir-221 5′-stem side mir start 367040TATGCCAGGTTCATG 2149 mir-221 mir-221 5′-stem side mir start 367041TTGTATGCCAGGTTC 2150 mir-221 mir-221 5′-stem side mir end 367042ATCTACATTGTATGC 2151 mir-221 mir-221 5′-stem side mir end 367043GAAATCTACATTGTA 2152 mir-221 mir-221 5′-stem side mir end 367044ACAGAAATCTACATT 2153 mir-221 mir-221 5′-stem side mir end 367045AACACAGAAATCTAC 2154 mir-221 mir-221 loop start 367046 CTGTTGCCTAACGAA2155 mir-221 mir-221 loop start 367047 AGCTGTTGCCTAACG 2156 mir-221mir-221 loop start 367048 GTAGCTGTTGCCTAA 2157 mir-221 mir-221 loop end367049 GAACACAGAAATCTA 2158 mir-221 mir-221 loop end 367050ACGAACACAGAAATC 2159 mir-221 mir-221 loop end 367051 TAACGAACACAGAAA2160 mir-221 mir-221 loop end 367052 CCTAACGAACACAGA 2161 mir-221mir-221 loop end 367053 TGCCTAACGAACACA 2162 mir-221 mir-221 3′-stemside mir start 367054 AATGTAGCTGTTGCC 2163 mir-221 mir-221 3′-stem sidemir start 367055 GACAATGTAGCTGTT 2164 mir-221 mir-221 3′-stem side mirstart 367056 GCAGACAATGTAGCT 2165 mir-221 mir-221 3′-stem side mir end367057 AAACCCAGCAGACAA 2166 mir-221 mir-221 3′-stem side mir end 367058AGCCTGAAACCCAGC 2167 mir-221 mir-221 3′-stem side mir end 367059GGTAGCCTGAAACCC 2168 mir-221 mir-221 3′-stem side mir end 367060CCAGGTAGCCTGAAA 2169 mir-221 mir-221 3′-stem side mir end 367061TTTCCAGGTAGCCTG 2170

These modified oligomeric compounds targeting pri-miRNAs can betransfected into preadipocytes or other undifferentiated cells, whichare then induced to differentiate, and it can be determined whetherthese modified oligomeric compounds act to inhibit or promote cellulardifferentiation. These compounds can be transfected into differentiatingadipocytes and their effects on expression levels of the pri-miRNAmolecules assessed in pre-adipocytes vs. differentiated adipocytes. Byusing a primer/probe set specific for the pri-miRNA or the pre-miRNA,real-time RT-PCR methods can be used to determine whether modifiedoligomeric compounds targeting pri-miRNAs can affect the expression orprocessing of the mature miRNAs from the pri-miRNA or pre-miRNAmolecules.

Example 39 Effects of Oligomeric Compounds Targeting miRNAs in theImmune Response

To investigate the role of miRNAs in the immune response, oligomericcompounds of the present invention targeting miRNAs were tested fortheir effects upon lipopolysaccharide (LPS)-activated primary murinemacrophages. Macrophages participate in the immune response, forexample, in the recognition and phagocytosis of microorganisms,including bacteria. Interferon-gamma (IFN-gamma) released by helper Tcells is one type of signal required for macrophage activation, and LPScan serve as an additional stimulus. LPS is a component of thegram-negative bacterial cell wall and acts as an agonist for toll-likereceptor 4 (TLR4), the primary LPS receptor expressed by macrophages.The proinflammatory cytokines interleukin-12 (IL-12) and interleukin-6(IL-6) are induced by LPS treatment of macrophages, thus the expressionof the mRNAs encoding these cytokines was used to evaluate the responseof macrophages to LPS following treatment with oligomeric compoundstargeting miRNAs.

Macrophages were isolated as follows. Female C57Bl/6 mice (Charles RiverLaboratories, Wilmington, Mass.) were injected intraperitoneally with 1ml 3% thioglycollate broth (Sigma-Aldrich, St. Louis, Mo.), andperitoneal macrophage cells were isolated by peritoneal lavage 4 dayslater. The cells were plated in 96-well plates at 40,000 cells/well forone hour in serum-free RPMI adjusted to contain 10 mM HEPES (InvitrogenLife Technologies, Carlsbad, Calif.), allowed to adhere, thennon-adherent cells were washed away and the media was replaced with RPMIcontaining 10 mM HEPES, 10% FBS and penicillin/streptomycin (“complete”RPMI; Invitrogen Life Technologies, Carlsbad, Calif.).

Oligomeric compounds were introduced into the cells using thenon-liposomal transfection reagent FuGENE 6 Transfection Reagent (RocheDiagnostics Corp., Indianapolis, Ind.). Oligomeric compound was mixedwith FuGENE 6 in 1 mL of serum-free RPMI to achieve a concentration of10 μL FuGENE per 1000 nM oligomeric compound. The oligomericcompound/FuGENE complex was allowed to form at room temperature for 20minutes. This mixture was diluted to the desired concentration ofoligomeric compound by the addition of the appropriate volume ofcomplete RPMI. The final ratio of FuGENE 6 to oligomeric compound was 1μL of FuGENE 6 per 100 nM oligomeric compound. A volume of 100 μL ofoligomeric compound/FuGENE/RPMI was added to each well of the 96-wellplate in which the macrophages were cultured. Each oligomeric compoundtreatment was repeated in triplicate.

Following oligomeric compound treatment, cells were stimultated withLPS. Cells were cultured in the presence of the transfection complex forapproximately 24 to 28 hours at 37° C. and 5% CO₂, after which themedium containing the transfection complex was removed from the cells,and complete RPMI containing 100 ng/mL LPS (Sigma-Aldrich Corp., St.Louis, Mo.) was added to the cells for a period of approximately 24hours. Control samples included (1) cells receiving no oligomericcompound, stimulated with LPS and (2) cells receiving neither oligomericcompound nor LPS treatment.

In another embodiment, following oligomeric compound treatment, cellswere first activated by IFN-gamma, to amplify the response to LPS. Cellswere cultured in the presence of the transfection complex forapproximately 24 hours at 37° C. and 5% CO₂, at which point the mediumcontaining the transfection complex was removed from the cells, andcomplete RPMI containing 100 ng/mL recombinant mouse IFN-gamma (R&DSystems, Minneapolis, Minn.) was added to the cells. After the 4 hourtreatment with INF-gamma, cells were treated with 100 ng/mL LPS forapproximately 24 hours. Control samples included (1) cells receiving nooligomeric compound, stimulated with LPS and (2) cells receiving neitheroligomeric compound nor LPS treatment.

Oligomeric compounds used as negative controls included ISIS 129690 (SEQID NO: 907), a universal scrambled control; ISIS 342673 (SEQ ID NO:758), an oligomeric compound containing 15 mismatches with respect tothe mature mir-143 miRNA; ISIS 342683 (SEQ ID NO: 790), an oligomericcompound representing the scrambled nucleotide sequence of an unrelatedPTP1B antisense oligonucleotide; and ISIS 289606 (CCTTCCCTGAAGGTTCCTCC,incorporated herein as SEQ ID NO: 863), an oligomeric compoundrepresenting the scrambled nucleotide sequence of an unrelated PTP1Bantisense oligonucleotide. ISIS 289606 is uniformly composed of 2′-MOEnucleotides, with phosphorothioate internucleoside linkages throughoutthe compound. All cytidines are 5-methyl cytidines. Used as a positivecontrol was ISIS 229927 (CCACATTGAGTTTCTTTAAG, incorporated herein asSEQ ID NO: 2171), targeting the mouse toll-like receptor 4 (TLR4) mRNA,which is the primary LPS receptor on macrophages. ISIS 229927 is achimeric oligomeric compound (“gapmer”) composed of a central “gap”region consisting of ten 2′-deoxynucleotides, which is flanked on bothsides (5′ and 3′ directions) by five nucleotide “wings,” wherein thewings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides.Internucleoside linkages are phosphorothioate throughout the compound,and all cytidines are 5-methylcytidines. Treatments with controloligomeric compounds were performed as described for oligomericcompounds targeting miRNAs.

Following the 24 hour treatment with LPS, the cells were lysed and RNAwas isolated using the RNEASY 96™ kit, as described herein. mRNAexpression was quantitated by real-time PCR, performed as describedherein, using primer and probe sets to amplify and quantitate TLR4,IL-12 and IL-6 mRNA expression levels. Primers and probe for TLR4,designed using GenBank Accession number NM_(—)021297.1, were: forwardprimer, 5′-CATGGAACACATGGCTGCTAA-3′ (SEQ ID NO: 2172), reverse primer,5′-GGAAAGGAAGGTGTCAGTGCTACT-3′ (SEQ ID NO: 2173), probe5′-FAM-TAGCATGGACCTTACCGGGCAGAAGG-TAMRA-3′ (SEQ ID NO: 2174). Primersand probe for IL-12, designed using GenBank Accession number M86671.1,were: forward primer, 5′-GCCAGTACACCTGCCACAAA-3′ (SEQ ID NO: 2175),reverse primer, 5′-GACCAAATTCCATTTTCCTTCTTG-3′ (SEQ ID NO: 2176), probe5′-FAM-AGGCGAGACTCTGAGCCACTCACATCTG-TAMRA-3′ (SEQ ID NO: 2177). Primersand probe for IL-6, designed using GenBank Accession number X54542.1,were: forward primer, 5′-CCTAGTGCGTTATGCCTAAGCA-3′ (SEQ ID NO: 2178),reverse primer, 5′-TTCGTAGAGAACAACATAAGTCAGATACC-3′ (SEQ ID NO: 2179),probe 5′-FAM-TTTCTGACCACAGTGAGGAATGTCCACAA-TAMRA-3′ (SEQ ID NO: 2180).The amount of total RNA in each sample was determined using a RibogreenAssay (Molecular Probes, Eugene, Oreg.), and expression levels of TLR4,IL-12 and IL-6 were normalized to total RNA.

TLR4 is the primary macrophage receptor for LPS. Thus, ISIS Number229927, targeted to TLR4, was tested for its ability to inhibit TLR4expression and interfere with the response of macrophages to LPS, bothwith and without pretreatment with IFN-gamma. The treatment of primarymurine macrophages with ISIS Number 229927 at reduced the expression ofTLR4 in a dose-dependent manner, in both LPS-stimulated and LPS- andIFN-gamma-stimulated cells. As judged by the dose-dependent reduction inIL-12, the response of macrophages to LPS was reduced followinginhibition of the TLR4 receptor expression, in both LPS-stimulated andLPS- and IFN-gamma-stimulated cells. These results demonstrated thatISIS 229927 can be used as a positive control for the inhibition ofIL-12 expression in macrophages responding to LPS.

Primary mouse macrophages were treated with a selected group ofoligomeric compounds targeting various miRNAs. These compounds and theirmiRNA targets are shown in Table 72. Table 72 shows IL-12 mRNAexpression following treatment with 300 nM of oligomeric compounds andLPS (−IFN), and IL-12 mRNA expression following treatment with 300 nM ofoligomeric compounds and stimulation with IFN-gamma and LPS (+IFN). The“−IFN” data represents a single experiment, and the “+IFN” datarepresents the average of 2 experiments. Data were normalized to valuesfrom cells receiving no oligomeric compound that were treated with LPS.IL-12 expression in cells receiving neither oligomeric compound nor LPStreatment was 2% of the control, both with and without IFN-gammapretreatment, demonstrating that IL-12 mRNA expression was notstimulated in the absence of LPS treatment. Where present, “N.D.”indicates “not determined”.

TABLE 72 IL-12 mRNA expression in primary macrophages treated witholigomeric compounds targeting miRNAs and stimulated with LPS ISIS SEQID −IFN +IFN NO: NO: pri-miRNA % UTC % UTC 289606 863 Scrambled controlN.D. 129 342673 758 mismatch to mir-143 91 N.D. 129690 907 Universalcontrol 73 129 229927 2171 TLR4 92 145 327874 292 mir-30a 202 15 327876294 mir-29b-1 194 9 327883 301 mir-27b 266 39 327887 305 mir-132 287 33327889 307 mir-23b 153 10 327890 308 let-7i 183 94 327893 311 let-7b 11752 327899 317 mir-183 164 7 327901 319 mir-143 225 9 327903 321 let-7a-3200 23 327912 330 let-7f-1 206 39 327913 331 mir-29c 276 73 327917 335mir-21 225 35 327919 337 mir-221 179 37 327920 338 mir-222 171 68 327921339 mir-30d 325 24 327923 341 mir-128b 269 134 327924 342 mir-129-2 17188 327925 343 mir-133b 302 60 327927 345 mir-15b 164 33 327928 346mir-29a-1 201 61 327931 349 let-7c 105 48 327935 353 mir-20 254 24327936 354 mir-133a-1 221 55 327940 358 mir-199a-2 228 107 327941 359mir-181b 89 34 327945 363 mir-24-2 202 68 327956 374 mir-216 212 59327958 376 mir-187 188 60 327959 377 mir-210 183 20 327961 379 mir-223203 10 327963 381 mir-26b 224 23 327967 385 let-7g 203 43 327971 389mir-23a 146 17 328105 407 hypothetical miRNA-088 108 57 328110 412hypothetical miRNA-107 221 8 328117 419 hypothetical miRNA-144 162 72328123 425 hypothetical miRNA-166 176 14 328129 431 hypotheticalmiRNA-173 87 10 328133 435 hypothetical miRNA-178 165 62 328137 439hypothetical miRNA-183 213 12 328138 440 hypothetical miRNA-185 277 31340341 236 mir-104 (Mourelatos) 139 13 340345 1882 miR-27 (Mourelatos)104 78 341786 1845 miR-149 266 99 341790 1843 miR-154 318 84 341793 1836miR-142-as 202 147 341800 1766 miR-186 180 100 341811 1906 miR-194 15488 341815 1831 miR-200a 190 157

A comparison of the data from IFN-gamma-stimulated and unstimulatedcells reveals that many of the oligomeric compounds targeting miRNAsattenuated the response of macrophages to LPS, as judged by IL-12 mRNAexpression, when the cells were activated with IFN-gamma prior to LPStreatment. When macrophages were pretreated with IFN-gamma, treatmentwith several of the oligomeric compounds, such as ISIS Number 328110,ISIS Number 327901, ISIS Number 327899, ISIS Number 327876 and ISISNumber 327961 resulted in a reduction in IL-12 mRNA expression rangingfrom 20-fold to 30-fold. Other oligomeric compounds, such as ISIS Number341800, ISIS Number 341811, ISIS Number 341793, ISIS Number 340345 andISIS Number 341815 resulted in a less pronounced reduction in IL-12 mRNAexpression ranging from 1.2-fold to 2-fold.

In a further embodiment, oligomeric compounds ISIS Number 327941targeting mir-181b and ISIS Number 327921 targeting mir-30d wereselected for a dose response study in LPS-stimulated primarymacrophages, with and without IFN-gamma pre-treatment. Cells weretreated as described herein, with oligomeric compound doses of 75, 150,300 and 600 nM. Untreated control cells received no oligomeric compoundtreatment but did receive LPS treatment. ISIS 229927 (SEQ ID NO: 2171)was used as a positive control and ISIS 342683 (SEQ ID NO: 790), ISIS126690 (SEQ ID NO: 907) and ISIS 289606 (SEQ ID NO: 863) were used asnegative controls. IL-12 and IL-6 mRNA expression levels were measuredby real-time PCR and normalized to untreated control cells that receivedLPS treatment. The IL-12 expression data, shown in Table 73, representthe average of 3 treatments. In cells receiving neither oligomericcompound nor LPS treatment, IL-12 expression was undetectable inIFN-gamma stimulated cells and was 1% of the untreated control inunstimulated cells.

TABLE 73 IL-12 mRNA expression following treatment of primary mousemacrophages with oligomeric compounds targeting mir-181b and mir-30d andLPS: dose response study IL-12 mRNA expression, % UTC SEQ Dose ofoligomeric compound ISIS ID 75 nM 150 nM 300 nM 600 nM NO: NO: −IFN +IFN−IFN +IFN −IFN +IFN −IFN +IFN 327941 359 49 4 45 2 34 3 41 3 327921 339109 14 88 7 67 5 53 5 229927 2171 67 46 53 35 45 16 46 8 342683 790 12192 165 76 147 65 130 64 129690 907 114 66 109 54 101 66 128 81 289606863 89 59 99 46 80 52 98 66

These data reveal that ISIS Number 327941 inhibited IL-12 expression incells stimulated with LPS alone, where the percentage of untreatedcontrol ranged from 34% to 49%. ISIS Number 327921 inhibited IL-12 mRNAexpression in a dose-dependent manner in cells stimulated with LPSalone, with the lowest IL-12 expression at 53% of untreated control. Incells pretreated with IFN-gamma and subsequently treated with LPS, ISISNumber 327941 markedly reduced IL-12 mRNA expression to less than 5% ofthe untreated control at all doses. ISIS Number 327921 reduced IL-12expression to 14% of the control at all 75 nM and to less than 10% ofthe untreated control at all other doses. Thus, ISIS Number 327941,targeting mir-181b, and ISIS Number 327921, targeting mir-30d, resultedin a greater reduction in IL-12 expression than ISIS 229927, which istargeted to TLR4.

The IL-6 expression data, shown in Table 74, represents the average of 3treatments. In cells receiving neither oligomeric compound nor LPStreatment, IL-12 expression was undetectable in IFN-gamma stimulatedcells and was 2% of the untreated control in unstimulated cells.

TABLE 74 IL-6 mRNA expression following treatment of primary mousemacrophages with oligomeric compounds targeting mir-181b and mir-30d andLPS: dose response study IL-6 mRNA expression, % UTC SEQ Dose ofoligomeric compound ISIS ID 75 nM 150 nM 300 nM 600 nM NO: NO: −IFN +IFN−IFN +IFN −IFN +IFN −IFN +IFN 327941 359 293 181 325 197 271 197 501 301327921 339 223 122 294 144 522 287 632 313 229927 2171 57 54 52 39 44 40104 69 342683 790 135 115 161 86 156 110 311 149 129690 907 98 92 99 86109 94 258 203 289606 863 77 78 68 69 65 70 77 59

These data reveal that, in contrast to IL-12 expression, IL-6 expressionis increased in a dose-dependent manner following treatment with ISISNumber 327941 and ISIS Number 327921, in both IFN-gamma-stimulated andunstimulated cells. This is in contrast to treatment with ISIS 229927,which exhibited some reduction in IL-6 expression in bothIFN-gamma-stimulated and unstimulated cells.

Abnormalities in the signaling pathways controlling the expression ofcytokines and cytokine receptors have been implicated in a number ofdiseases. Compounds that modulate the activity of macrophages, forexample, the response to foreign antigens such as LPS, are candidatetherapeutic agents with application in the treatment of conditionsinvolving macrophage activation, such as septic shock and toxic shock

The expression of mir-181 in mouse cells and tissues was evaluated byNorthern blot. Mouse tissues RNA was purchased from Ambion, Inc.(Austin, Tex.). RNA was prepared from macrophages were prepared andstimulated with LPS as described herein. Northern blotting was performedas described herein, and mir-181 levels were normalized to U6 levels,both of which were quantitated by Phosphorimager analysis. Expressionlevels are presented in arbitrary units. mir-181 was found to be mosthighly expressed in lung and kidney, at approximately equal levels. Thenext highest expression levels were found in brain, heart and liver. Forexample, as compared to kidney mir-181 expression levels, mir-181 wasexpressed approximately 2.5-fold lower in brain, approximately 2.2-foldlower in heart and approximately 1.8-fold lower in liver. mir-181 levelsin both naïve and LPS-stimulated macrophages were 4.5-fold and 4.9-foldlower than in kidney, respectively. The lowest expression levels werefound in thymus and spleen, which were 12.9-fold and 14.7-fold less ascompared to kidney.

Example 40 Adipocyte Assay of Oligomeric Compounds

The effect of several oligomeric compounds of the present inventiontargeting miRNA target nucleic acids on the expression of markers ofcellular differentiation was examined in differentiating adipocytes.

As described in Example 13, some genes known to be upregulated duringadipocyte differentiation include HSL, aP2, Glut4 and PPARγ. These genesplay important roles in the uptake of glucose and the metabolism andutilization of fats. An increase in triglyceride content is anotherwell-established marker for adipocyte differentiation.

For assaying adipocyte differentiation, expression of the four hallmarkgenes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG)accumulation were measured as previously described in adipocytestransfected with oligomeric compounds targeting miRNAs. Triglyceridelevels as well as mRNA levels for each of the four adipocytedifferentiation hallmark genes are expressed as a percentage ofuntreated control (UTC) levels. In this experiment, the negative controloligomeric compound was ISIS Number 342672 (SEQ ID NO: 789) or ISISNumber 342673 (SEQ ID NO: 758). Results are shown in Table 75. Eachvalue represents at least one oligomeric compound treatment; data frommore than one oligomeric compound treatment were averaged. Wherepresent, “N.D.” indicates “not determined”.

TABLE 75 Effects of oligomeric compounds targeting miRNAs on expressionof adipocyte differentiation markers SEQ Isis ID PPAR Number NOPri-miRNA TG HSL aP2 GLUT4 gamma UTC N/A N/A 100 100 100 100 100 327873291 mir-140 105 116 113 106 104 327879 297 mir-7-1/mir-7-1* 59 103 10399 81 327881 299 mir-128a 91 93 95 97 98 327885 303 mir-17/mir-91 29 5769 40 59 327886 304 mir-123/mir-126 12 22 19 13 25 327887 305 mir-132 5453 60 43 81 327891 309 mir-212 22 52 56 47 50 327895 313 mir-122a 76 8890 76 86 327896 314 mir-22 22 37 43 35 52 327897 315 mir-92-1 28 39 6232 66 327898 316 mir-142 102 92 96 82 101 327899 317 mir-183 25 27 47 1462 327900 318 mir-214 26 21 32 12 55 327902 320 mir-192-1 55 56 58 15 56327906 324 mir-103-1 25 37 46 14 50 327907 325 mir-26a-1 19 21 29 6 49327910 328 mir-107 24 32 35 16 39 327911 329 mir-106 59 71 76 48 75327912 330 let-7f-1 112 95 101 79 78 327916 334 mir-124a-2 56 64 67 5171 327917 335 mir-21 26 26 32 15 54 327918 336 mir-144 65 85 91 66 74327920 338 mir-222 20 14 22 0 34 327921 339 mir-30d 56 76 76 36 75327923 341 mir-128b 88 64 65 54 77 327929 347 mir-199b 65 68 62 49 71327935 353 mir-20 41 61 60 47 67 327936 354 mir-133a-1 23 40 40 6 47327940 358 mir-199a-2 62 67 62 43 64 327943 361 mir-18 112 109 106 87 98327944 362 mir-220 38 55 71 28 64 327945 363 mir-24-2 48 41 43 26 51327946 364 mir-211 82 76 73 68 81 327949 367 mir-10a 43 49 52 20 54327950 368 mir-19a 125 94 95 104 93 327952 370 mir-137 93 64 56 61 84327957 375 mir-100-1 29 15 23 11 68 327958 376 mir-187 28 5 10 5 55327959 377 mir-210 33 11 24 152 65 327961 379 mir-223 77 88 91 101 95327962 380 mir-30c-1 64 77 75 58 80 327963 381 mir-26b 124 89 75 91 91327964 382 mir-152 60 102 96 114 93 327965 383 mir-135-1 116 84 67 88 91327966 384 mir-217 52 56 53 43 77 327968 386 sterol regulatory 94 79 6785 79 element-binding protein-1/mir-33b 327969 387 mir-182 34 45 44 3667 327970 388 mir-148a 48 25 29 27 46 327971 389 mir-23a 45 38 49 60 69327972 390 mir-181c 67 70 70 75 85 328089 391 hypothetical miR- 67 55 5059 79 13/miR-190 328090 392 hypothetical miRNA-023 128 81 68 86 95328091 393 hypothetical miRNA-30 48 40 46 26 85 328092 394 glutamatereceptor, 134 80 74 78 86 ionotrophic, AMPA 3/ hypothetical miRNA-033328094 396 hypothetical miRNA-040 65 74 68 83 94 328095 397 hypotheticalmiRNA-041 110 83 70 98 92 328096 398 hypothetical miRNA-043 74 76 71 7989 328097 399 hypothetical miRNA-044 65 54 48 62 63 328098 400hypothetical miRNA-055 39 28 23 25 54 328099 401 hypothetical miRNA-05857 74 80 61 72 328100 402 hypothetical miRNA-070 20 49 47 39 48 328101403 LOC 114614 containing 67 78 83 57 70 miR-155/hypothetical miRNA-071328102 404 hypothetical miRNA-075 70 99 96 58 94 328103 405 hypotheticalmiRNA-079 113 87 96 86 83 328104 406 hypothetical miRNA-083 64 81 94 8373 328105 407 DiGeorge syndrome 82 95 102 75 85 critical region gene8/hypothetical miRNA-088 328106 408 hypothetical miRNA-090 70 86 91 7981 328107 409 hypothetical miRNA-099 51 55 68 52 71 328108 410hypothetical miRNA-101 79 75 87 65 72 328109 411 hypothetical miRNA-10523 62 68 55 69 328110 412 hypothetical miRNA-107 96 84 89 77 80 328111413 hypothetical miRNA-111 65 77 79 50 65 328113 415 hypotheticalmiRNA-137 74 83 87 78 85 328115 417 hypothetical miRNA-142 53 75 74 8480 328116 418 hypothetical miRNA-143 107 91 99 105 95 328117 419collagen, type I, 16 18 28 13 42 alpha 1/hypothetical miRNA-144 328118420 hypothetical miRNA-153 69 67 74 57 72 328119 421 hypotheticalmiRNA-154 109 101 119 104 102 328120 422 hypothetical miRNA-156 80 67 8068 73 328121 423 hypothetical miRNA-161 119 110 119 115 105 328122 424hypothetical miRNA-164 97 89 99 91 103 328123 425 hypothetical miRNA-16654 91 119 129 88 328124 426 hypothetical miRNA- 108 96 118 105 92168-1/similar to ribosomal protein L5 328125 427 forkhead box 44 48 7565 68 P2/hypothetical miRNA-169 328126 428 hypothetical miRNA-170 108135 120 107 98 328127 429 glutamate receptor, 81 93 95 75 85 ionotropic,AMPA 2/ hypothetical miRNA-171 328128 430 hypothetical miRNA-172 61 7290 73 86 328129 431 hypothetical miRNA-173 19 34 54 36 59 328130 432hypothetical miRNA-175 91 64 72 55 77 328131 433 hypothetical miRNA-17674 51 63 56 55 328133 435 hypothetical miRNA-178 43 49 66 59 53 328134436 hypothetical miRNA-179 107 109 97 109 86 328135 437 cezanne 2/ 29 2034 19 33 hypothetical miRNA-180 328136 438 hypothetical miRNA-181 26 3757 35 54 328137 439 tight junction protein 37 25 45 29 36 1 (zonaoccludens 1)/ hypothetical miRNA-183 328138 440 hypothetical miRNA-18580 56 52 52 63 328139 441 hypothetical miRNA-188 90 116 100 85 91 340341236 mir-104 (Mourelatos) 46 49 62 48 71 340343 1780 mir-105 (Mourelatos)35 46 60 33 59 340348 848 mir-93 (Mourelatos) 48 57 68 52 78 340350 855mir-95 (Mourelatos) 38 45 64 53 59 340352 1821 mir-99 (Mourelatos) 110123 107 97 102 340354 1903 mir-25 64 56 72 61 74 340356 1853 mir-28 4359 73 54 62 340358 1825 mir-31 23 24 47 21 42 340360 1865 mir-32 106 102102 91 96 341791 1880 mir-30a 50 72 80 47 75 341795 1762 mir-199a-2 5774 76 55 74 341796 1904 mir-131-1/mir-9 59 67 74 58 66 341797 1773mir-17/mir-91 20 29 45 17 50 341798 1871 mir-123/mir-126 62 77 84 55 70341799 1787 hypothetical miR- 98 103 101 89 89 13/miR-190 341800 1766mir-186 18 42 50 28 61 341801 1839 mir-198 65 89 90 77 82 341802 1806mir-191 155 121 98 85 127 341803 760 mir-206 N.D. 79 85 73 68 341804 761mir-94/mir-106b N.D. 75 78 62 71 341805 762 mir-184 N.D. 86 90 74 77341806 763 mir-195 N.D. 77 83 58 70 341807 764 mir-193 N.D. 102 82 10183 344268 1774 mir-10b 57 44 46 22 53 344269 1890 mir-29c 42 35 41 28 48344275 1912 mir-203 36 39 36 21 46 344276 1828 mir-204 66 68 72 49 72344277 1767 mir-1d-2 75 57 61 45 68 344338 1812 mir-130a 103 89 86 66 91344340 1921 mir-140 60 47 82 16 67 344341 1823 mir-218-1 50 33 42 14 49344342 1814 mir-129-2 88 87 88 71 83 344343 1811 mir-130b 32 22 25 4 30344611 1785 mir-240* (Kosik) 43 31 34 3 34 344612 1790 mir-232* (Kosik)69 59 72 40 62 344613 1775 mir-227* (Kosik)/mir- 47 46 55 38 57 226*(Kosik) 344614 1834 mir-227* (Kosik)/mir- 89 71 78 61 86 226* (Kosik)344615 1900 mir-244* (Kosik) 149 154 166 145 144 344616 1800 mir-224*(Kosik) 32 23 26 2 36 344617 1862 mir-248* (Kosik) 52 55 59 42 72 3466851884 mir-27 (Mourelatos) 164 172 181 233 138 346686 1857 mir-101-1 73 8083 73 83 346687 1802 mir-129-1 55 53 56 35 60 346688 1898 mir-182 33 3948 12 55 346689 1830 mir-200b 59 63 79 45 64 346691 1870 mir-147(Sanger) 56 69 69 64 79 346692 1889 mir-224 (Sanger) 35 18 26 11 28346693 1838 mir-134 (Sanger) 69 66 77 65 81 346694 1763 mir-146 (Sanger)31 18 41 5 32 346695 1824 mir-150 (Sanger) 69 73 72 58 78 346906 1781mir-296 (RFAM/mmu) 83 70 77 70 80 346907 1815 mir-299 (RFAM/mmu) 47 3650 37 51 346908 1881 mir-301 (RFAM/mmu) 75 71 77 65 77 346909 1902mir-302 (RFAM/mmu) 66 64 68 64 77 346910 1866 mir-34a (RFAM/mmu) 80 6978 63 83 346913 1795 let-7d 63 58 66 40 59 346914 1810 mir-94/mir-106b41 27 48 16 41 346915 1784 mir-200a 73 67 83 75 90 346917 1826 mir-31 3927 33 20 31 346919 1849 mir-93 (Mourelatos) 44 45 64 50 65 346920 1801mir-96 63 53 70 61 70 346921 1759 mir-34 52 49 69 51 62 348116 1922mir-320 43 58 79 48 76 348117 1860 mir-321-1 66 55 70 73 65 348119 1908mir-142 91 76 81 86 90 348124 1820 mir-10b 53 43 59 41 63 348125 1878mir-19b-1 79 64 67 65 64 348127 1869 mir-27b 155 150 185 201 130

Several compounds were found to have effects on adipocytedifferentiation. For example, the oligomeric compounds ISIS Number340348 (SEQ ID NO: 848), targeted to mir-93 (Mourelatos); ISIS Number341798 (SEQ ID NO: 1871), targeted to mir-123/mir-126; ISIS Number344340 (SEQ ID NO: 1921) targeted to mir-140; ISIS Number 346687 (SEQ IDNO: 1802), targeted to mir-129-1 and ISIS Number 348117 (SEQ ID NO:1860), targeted to mir-321-1 were shown to significantly reduce theexpression levels of 3 of the 5 markers of adipocyte differentiation.The effects of ISIS Number 327897 (SEQ ID NO: 315), targeted tomir-92-1, were even more pronounced, as shown by the significantreduction in expression of 4 of the 5 markers of differentiation. Thesedata indicate that these oligomeric compounds have the ability to blockadipocyte differentiation. Therefore, these oligomeric compounds may beuseful as pharmaceutical agents with applications in the treatment,attenuation or prevention of obesity, hyperlipidemia, atherosclerosis,atherogenesis, diabetes, hypertension, or other metabolic diseases aswell as having potential applications in the maintenance of thepluripotent phenotype of stem or precursor cells.

Other compounds were shown to stimulate adipocyte differentiation. Forexample, the oligomeric compounds ISIS Number 328121 (SEQ ID NO: 423),targeted to hypothetical miRNA-161; ISIS Number 344615 (SEQ ID NO:1900), targeted to mir-244* (Kosik); ISIS Number 346685 (SEQ ID NO:1884), targeted to mir-27 (Mourelatos); and ISIS Number 348127 (SEQ IDNO: 1869), targeted to mir-27b resulted in significant increases in all5 markers of adipocyte differentiation. Other oligomeric compounds, forexample ISIS Number 340352 (SEQ ID NO: 1821), targeted to mir-99(Mourelatos) and ISIS Number 328126 (SEQ ID NO: 428), targeted tohypothetical miRNA-170, resulted in increases in 4 of the 5 markers ofadipocyte differentiation. These oligomeric compounds may be useful as apharmaceutical agents in the treatment of diseases in which theinduction of adipocyte differentiation is desirable, such as anorexia,or for conditions or injuries in which the induction of cellulardifferentiation is desirable, such as Alzheimers disease or centralnervous system injury, in which regeneration of neural tissue (such asfrom pluripotent stem cells) would be beneficial. Furthermore, thisoligomeric compound may be useful in the treatment, attenuation orprevention of diseases in which it is desirable to induce cellulardifferentiation and/or quiescence, for example in the treatment ofhyperproliferative disorders such as cancer.

In a further embodiment, oligomeric compounds of the present inventionwere tested for their effects on insulin signaling in HepG2 cells. Asdescribed in Example 18, insulin is known to regulate the expression ofhepatic IGFBP-1, PEPCK-c and follistatin. Thus, the IGFBP-1, PEPCK-c andfollistatin genes serve as marker genes for which mRNA expression can bemonitored and used as an indicator of an insulin-resistant state.Oligomeric compounds with the ability to reduce expression of IGFBP-1,PEPCK-c and follistatin are highly desirable as agents potentiallyuseful in the treatment of diabetes and hypertension oligomericcompounds of the invention were tested for their effects on insulinsignalling in liver-derived cells. For assaying insulin signalling,expression of IGFBP-1, PEPCK-c and follistatin mRNAs were measured aspreviously described in HepG2 cells transfected with oligomericcompounds targeting miRNAs and treated with either no insulin (“basal”Experiment 1, for identification of insulin-mimetic compounds) or with 1nM insulin (“insulin treated” Experiment 2, for identification ofinsulin sensitizers) for four hours. At the end of the insulin orno-insulin treatment, total RNA was isolated and real-time PCR wasperformed on all the total RNA samples using primer/probe sets for threeinsulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expressionlevels for each gene are normalized to total RNA, and values areexpressed relative to the transfectant only untreated control (UTC). Inthese experiments, the negative control oligomeric compound was ISISNumber 342672 (SEQ ID NO: 789) or ISIS Number 342673 (SEQ ID NO: 758).Results are shown in Tables 76 and 77. Each value represents at leastone oligomeric compound treatment; data from more than one oligomericcompound treatment were averaged.

TABLE 76 Experiment 1: Effects of oligomeric compounds targeting miRNAson insulin-repressed gene expression in HepG2 cells Isis SEQ ID NumberNO Pri-miRNA Follistatin IGFBP1 PEPCKc UTC N/A N/A 100 100 100 327873291 mir-140 97 108 72 327885 303 mir-17/mir-91 74 161 73 327886 304mir-123/mir-126 82 176 61 327887 305 mir-132 113 119 83 327893 311let-7b 93 107 81 327895 313 mir-122a 83 108 71 327897 315 mir-92-1 129163 72 327899 317 mir-183 66 105 42 327900 318 mir-214 111 102 88 327911329 mir-106 81 157 52 327916 334 mir-124a-2 108 102 88 327918 336mir-144 75 95 81 327920 338 mir-222 99 165 52 327923 341 mir-128b 86 11683 327946 364 mir-211 103 108 90 327949 367 mir-10a 112 112 81 327950368 mir-19a 83 109 65 327952 370 mir-137 93 123 70 327957 375 mir-100-169 143 59 327958 376 mir-187 91 119 73 327959 377 mir-210 98 124 139327961 379 mir-223 113 150 98 327963 381 mir-26b 101 108 92 327964 382mir-152 97 100 74 327965 383 mir-135-1 95 106 63 341800 1766 mir-186 105114 71 341801 1839 mir-198 85 99 73 341802 1806 mir-191 136 186 98341803 760 mir-206 68 107 110 341804 761 mir-94/mir-106b 63 162 44341805 762 mir-184 63 105 40 341806 763 mir-195 75 128 79 341807 764mir-193 102 129 97 341808 1861 mir-185 96 113 64

Under “basal” conditions (without insulin), treatments of HepG2 cellswith oligomeric compounds of the present invention resulting indecreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/orfollistatin marker genes indicate that the oligomeric compounds have aninsulin mimetic effect. Treatments with oligomeric compounds of thepresent invention resulting in an increase in mRNA expression levels ofthe PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that thesecompounds inhibit or counteract the normal insulin repression of mRNAexpression of these genes.

From these data, it is evident that the oligomeric compounds, ISISNumber 327886 (SEQ ID NO: 304), targeting mir-123/mir-126; ISIS Number327899 (SEQ ID NO: 317), targeting mir-183; ISIS Number 327911 (SEQ IDNO: 329), targeting mir-106; ISIS Number 327920 (SEQ ID NO: 338),targeting mir-222; ISIS Number 341804 (SEQ ID NO: 761), targetingmir-94/mir-106b; and ISIS Number 341805 (SEQ ID NO: 762), targetingmir-184, for example, resulted in 39%, 58%, 48%, 48%, 56% and 60%reductions, respectively, in PEPCK-c mRNA, a marker widely considered tobe insulin-responsive. Thus, these oligomeric compounds may be useful aspharmaceutic agents comprising insulin mimetic properties in thetreatment, amelioration, or prevention of diabetes or other metabolicdiseases.

Conversely, the results observed with the oligomeric compounds targetingmir-92-1 (ISIS Number 327897, SEQ ID NO: 315), mir-10a (ISIS Number327949, SEQ ID NO: 367), mir-223 (ISIS Number 327961, SEQ ID NO: 379)and mir-191 (ISIS Number 341802, SEQ ID NO: 1806), for example,exhibited increased expression of the IGFBP-1 and follistatin markergenes, suggesting that the mir-92-1, mir-10a, mir-223, and mir-191 miRNAtargets may be involved in the regulation of these insulin-responsivegenes. When these miRNAs are inactivated by an oligomeric compound,IGFBP-1 and follistatin gene expression is no longer repressed.Similarly, treatment oligomeric compounds targeting mir-210 (ISIS Number327959, SEQ ID NO: 377)) and mir-206 (ISIS Number 341803, SEQ ID NO:760) resulted in increases in the IGFBP-1 and PEPCK-c marker genes,suggesting that mir-210 and mir-206 may be involved in the regulation ofthese insulin-responsive genes.

TABLE 77 Experiment 2: Effects of oligomeric compounds targeting miRNAson insulin-sensitization of gene expression in HepG2 cells SEQ Isis IDNumber NO Pri-miRNA Follistatin IGFBP1 PEPCKc UTC + N/A N/A 100 100 1001 nM insulin 327897 315 mir-92-1 123 243 78 327911 329 mir-106 71 160 78327916 334 mir-124a-2 98 128 88 327918 336 mir-144 76 81 107 327920 338mir-222 102 267 59 327923 341 mir-128b 106 119 125 327946 364 mir-211109 138 99 327949 367 mir-10a 111 172 101 327950 368 mir-19a 89 124 82327952 370 mir-137 100 103 85 327957 375 mir-100-1 73 184 88 327958 376mir-187 112 149 106 327959 377 mir-210 92 141 156 327961 379 mir-223 128160 126 327963 381 mir-26b 95 111 94 327964 382 mir-152 114 121 122327965 383 mir-135-1 79 105 64 328114 416 hypothetical 81 177 41miRNA-138 328115 417 hypothetical 91 120 59 miRNA-142 328125 427forkhead box 107 216 77 P2/hypothetical miRNA-169 328342 451 mir-203 8898 39 328343 452 mir-7-1/mir-7-1* 139 135 69 328358 467 mir-123/mir-126106 165 93 328367 476 mir-212 107 141 85 328377 486 hypothetical 159 247182 miRNA-30 328396 505 mir-205 135 128 65 328397 506 mir-103-1 75 57 76328423 532 mir-19b-2 114 69 77 328649 558 mir-20 69 115 86 328702 611mir-10a 88 83 96 328761 670 hypothetical 53 193 64 miRNA-138 328764 673hypothetical 128 145 68 miRNA-142 328769 678 mir-26b 84 110 100 328774683 sterol regulatory 68 100 77 element-binding protein-1/mir-33b 328776685 forkhead box 114 86 125 P2/hypothetical miRNA-169

For HepG2 cells treated with 1 nM insulin, treatments with oligomericcompounds of the present invention resulting in a decrease in mRNAexpression levels of the PEPCK-c, IGFBP-1 and/or follistatin markergenes indicate that these compounds have an insulin sensitizationeffect. Treatments with oligomeric compounds of the present inventionresulting in an increase in mRNA expression levels of the PEPCK-c,IGFBP-1 and/or follistatin marker genes indicate that these compoundsinhibit or counteract the normal insulin response of repression of mRNAexpression of these genes.

From these data, it is evident that the oligomeric compounds, ISISNumber 327920 (SEQ ID NO: 338), targeting mir-222; ISIS Number 328114(SEQ ID NO: 416), targeting hypothetical miRNA-138; ISIS Number 328115(SEQ ID NO: 417), targeting hypothetical miRNA-142; and ISIS Number328342 (SEQ ID NO: 451) targeting mir-203, for example, were observed toresult in a 41%, a 59%, a 41% and a 61% reduction, respectively, ofPEPCK-c mRNA expression, widely considered to be a marker ofinsulin-responsiveness. Thus, these oligomeric compounds may be usefulas pharmaceutic agents with insulin-sensitizing properties in thetreatment, amelioration, or prevention of diabetes or other metabolicdiseases.

Conversely, the results observed with the oligomeric compounds targetingmir-128b (ISIS Number 327923, SEQ ID NO: 341), mir-223 (ISIS Number327961, SEQ ID NO: 379), mir-152 (ISIS Number 327964, SEQ ID NO: 382)and hypothetical miRNA-30 (ISIS Number 328377, SEQ ID NO: 486), allexhibiting increased expression of the IGFBP-1, PEPCK-c and follistatinmarker genes, support the conclusion that the mir-128b, mir-223, mir-152and hypothetical miRNA-30 may be involved in the regulation ofinsulin-responsive genes. When these miRNAs are inactivated by theoligomeric compounds of the present invention, IGFBP-1, PEPCK-c andfollistatin gene expression is no longer repressed or insulin-sensitive.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

What is claimed is:
 1. A method of inhibiting the activity of a microRNAcomprising contacting a cell comprising the microRNA with a compoundcomprising an oligomeric compound, wherein: the oligomeric compound isat least 90% complementary to a miRNA or a precursor thereof; theoligomeric compound consists of 15 to 30 linked monomeric subunits; atleast one monomeric subunit is a modified nucleoside; and the miRNA isselected from miR-33a (SEQ ID NO: 227) and miR-33b (SEQ ID NO: 286). 2.The method of claim 1 wherein the oligomeric compound is at least 95%complementary to the miRNA.
 3. The method of claim 1 wherein theoligomeric compound is 100% complementary to the miRNA.
 4. The method ofclaim 1 wherein the oligomeric compound is 100% complementary to atleast an 8-nucleobase portion of the miRNA.
 5. The method of claim 1wherein the miRNA is miR-33a (SEQ ID NO: 227).
 6. The method of claim 1wherein the miRNA is miR-33b (SEQ ID NO: 286).
 7. The method of claim 1wherein the oligomeric compound is 100% complementary to at least an8-nucleobase portion of miR-33a (SEQ ID NO: 227).
 8. The method of claim1 wherein the oligomeric compound is 100% complementary to at least an8-nucleobase portion of miR-33b (SEQ ID NO: 286).
 9. The method of claim1 wherein the oligomeric compound consists of an oligonucleotide. 10.The method of claim 9 wherein the oligonucleotide consists of 19 to 24linked nucleosides.
 11. The method of claim 1 wherein the oligomericcompound comprises at least one modified sugar moiety.
 12. The method ofclaim 1 wherein the modified sugar moiety is selected from a 2′-F sugarmoiety, a 2′-O-methyl sugar moiety, a 2′-O-methoxyethyl sugar moiety,and a bicyclic sugar moiety.
 13. The method of claim 12 wherein thebicyclic sugar moiety comprises a 4′-CH₂—O-2′ bridge.
 14. The method ofclaim 10 wherein the oligonucleotide comprises at least one modifiedsugar moiety.
 15. The method of claim 14 wherein the modified sugarmoiety is selected from a 2′-F sugar moiety, a 2′-O-methyl sugar moiety,a 2′-O-methoxyethyl sugar moiety, and a bicyclic sugar moiety.
 16. Themethod of claim 15 wherein the bicyclic sugar moiety comprises a4′-CH₂—O-2′ bridge.
 17. The method of claim 1 wherein the oligomericcompound comprises at least one modified internucleoside linkage. 18.The method of claim 17 wherein the modified internucleoside linkage is aphosphorothioate linkage.
 19. The method of claim 18 wherein eachinternucleoside linkage of the oligomeric compound is a phosphorothioatelinkage.
 20. The method of claim 1 wherein each monomeric subunitcomprises a modified sugar moiety.
 21. The method of claim 20 whereineach modified sugar moiety is independently selected from a 2′-F sugarmoiety, a 2′-O-methyl sugar moiety, a 2′-O-methoxyethyl sugar moiety,and a bicyclic sugar moiety.
 22. The method of claim 1 wherein theoligonucleotide comprises two or more chemically distinct regions. 23.The method of claim 22 wherein the sugar moiety of each nucleoside of afirst chemically distinct region is the same and is selected from a2′-fluoro nucleoside, a 2′-O-methyl nucleoside, a 2′-O-methoxyethylnucleoside, a 2′-deoxynucleoside, and a bicyclic sugar nucleoside. 24.The method of claim 22 wherein the sugar moiety of each nucleoside of afirst chemically distinct region is the same and is selected from a2′-deoxynucleoside and a bicyclic sugar nucleoside.
 25. The method ofclaim 24 wherein the bicyclic nucleoside comprises a 4′-CH₂—O-2′ bridge.26. The method of claim 1 wherein the oligomeric compound comprises atleast one modified nucleobase.
 27. The method of claim 26 wherein themodified nucleobase is 5-methylcytosine.
 28. A composition comprising acompound comprising an oligomeric compound, wherein: the oligomericcompound is at least 90% complementary to a miRNA or a precursorthereof; the oligomeric compound consists of 15 to 30 linked monomericsubunits; at least one monomeric subunit is a modified nucleoside; andthe miRNA is miR-33b (SEQ ID NO: 286); and a pharmaceutically acceptablediluent or carrier.